Human antibodies binding to ROR2

ABSTRACT

The present invention relates to fully human antibodies and conjugates thereof, which specifically bind to the extracellular domain of receptor tyrosine kinase-like orphan receptor 2 (ROR2).

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority under 35 U.S.C. § 371 of International Application No. PCT/EP2018/069826, filed on Jul. 20, 2018, which claims the benefit of priority of European Application No. 17182355.2, filed on Jul. 20, 2017. The contents of these earlier filed applications are hereby incorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING

The sequence listing submitted Jul. 27, 2020 as a text file named “13318_0048U1_Revised_Sequence_Listing,” created on Jul. 24, 2020, and having a size of 167,392 bytes, is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

FIELD OF THE INVENTION

The present invention relates to human antibodies binding to ROR2, preferably to human ROR2 (hROR2), and fragments and conjugates thereof, as well as to uses thereof.

BACKGROUND OF THE INVENTION

Cancer is one of the leading causes of death. It is a class of diseases caused by malignant transformation of healthy cells, resulting from genetic alterations, like chromosomal translocations, mutations in tumor suppressor genes, transcription factors or growth-factor receptors, leading to the immortalization of the cells. If the immortalization is combined with excessive proliferation, the immortalized cells generate tumors, with or without metastasis (in case of solid tumors), or leukemias and lymphomas (cancers of the blood). Defective apoptosis, or programmed cell death, can further contribute to malignant transformation of cells leading to cancer.

A family of membrane-associated receptor tyrosine kinases, consisting of the receptor tyrosine kinase orphan receptors-1 and -2 (ROR1 and ROR2) have been described as specifically associated with particular cancers (Rebagay et al. (2012) Front Oncol. 2(34):1-8; doi 10.3389/onc.2012.00034), while being largely absent in expression on healthy tissue with, a few exceptions e.g. in case of ROR1 (Balakrishnan et al. (2016) Clin Cancer Res. doi: 10.1158/1078-0432). Whether or not ROR expression is functionally associated with tumorigenesis remains unclear. However, due to the very tumor-selective expression of ROR family members, they represent relevant targets for targeted cancer therapies. Importantly, human ROR2 (hROR2) has been described to be expressed on tumor cells in neuroblastoma, sarcoma (and especially osteosarcoma), renal cell carcinoma, breast cancer, testicular cancer, ovarian cancer, pancreatic cancer, kidney cancer, renal cancer, gastric cancer, prostate cancer, head and neck cancer, melanoma, squamous cell carcinoma, multiple myeloma and other cancers.

Receptor tyrosine kinase orphan receptors-1 and -2, ROR1 and ROR2, are the only two family members defining a new receptor tyrosine kinase family, based on the overall structural design and some functional similarities. Both ROR1 and ROR2 proteins are type I-single pass trans-membrane receptors with an extracellular domain (ECD) consisting of an immunoglobulin domain, a cysteine rich frizzled domain and a Kringle domain. These three extracellular domains are followed by a trans-membrane domain connecting the ECD to an intracellular portion of the protein comprising kinase domains (Rebagay et al. (2012) Frontiers Oncol. 2: 1-8).

The human ROR1 and ROR2 proteins are 58% homologous between each other, but each of the ROR proteins is highly conserved between species. This represents a challenge for the development of human ROR2 specific monoclonal antibodies and very few antibodies are known.

In normal physiology, hROR2 is responsible for aspects of bone and cartilage growth during embryonic development. After birth, expression of hROR2 is downregulated and hROR2 is normally undetectable or expressed at very low levels in adult tissues. Weak expression of hROR2 has only been reported in stomach and thyroid issue (Morioka et al. (2009) Cancer Sci. 100: 1227-1233). hROR2 has previously been recognized as a target for the development of hROR2 specific antibodies (WO 2013/103637 A1, WO 2016/142768 A1.

There is a need for high-quality anti-ROR2 antibodies that can be used as a basis for the development of antibody-based targeted therapies of ROR2-expressing cancers. In particular, there is also a need for anti-ROR2 antibodies with excellent developability parameters, including a low propensity for aggregation, and that exhibit high thermal stability, which are typically obtained by B cells from immunized animals. Further, there is a need for antibodies that, in addition to binding human ROR2, also bind to ROR2 of standard toxicology species, like mice, rats, rabbits and/or cynomolgus monkeys, and especially to both cynomolgus and mouse ROR2, allowing toxicological characterization of antibody-based targeted therapies in anticipation of human clinical trials. Additionally, for human therapy there is a need for antibodies that are essentially fully human antibodies with lowest immunogenicity risk upon systemic administration in human subjects. There is also a need for additional diagnostic tools for detecting ROR2 expressions in ROR2-related disease conditions. The instant invention is directed to addressing these and other needs.

SUMMARY OF THE INVENTION

The present invention provides fully human antibodies which specifically bind to the extracellular domain of receptor tyrosine kinase-like orphan receptor 2 (ROR2). The invention and general advantages of its features will be discussed in detail below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of the process used to develop novel human antibodies targeting hROR2. (A) In a first step, human antibody transgenic mice (H2L2 mice) (described in WO 2010/070263 A1) were immunized with the entirely extracellular domain of C-terminally tagged with a Twin-Strep-tag (hROR2-TwinStrep) (B) Then, splenic B cells binding to hROR2-TwinStrep were enriched from immunized H2L2 mice by Magnetic Activated Cell Sorting (MACS). (C) From the isolated splenic B cells transposable expression vector libraries for human IgG1 were generated by PCR-cloning of variable heavy and light chain domains from the isolated hROR2-binding splenic B cells. (D) This was followed by transposition-mediated generation of cellular libraries displaying full-length human IgG1 libraries stably expressing the PCR-cloned antibody libraries in immortalized preB cells. (E) FACS-based screening of surface-displayed IgG1 antibodies for hROR2 binding and isolation of single cell clones by FACS. (F) Functional screening IgG1 antibodies of selected clonal supernatants. (G) PCR amplification of antibodies from selected clones.

FIG. 2. anti-hROR2-ECD antibody titer detection following immunization of H2L2 mice referred to as 1357, 1368 and 1359 with ECD-hROR2-TwinStrep. Data for pre-immunization, and on days 7, 28 and 49 following initial immunization are shown.

FIG. 3. Antigen-specific FACS sorting per mouse based on IgG/IgK and IgM/IgK libraries for H2L2 mouse 1357, H2L2 mouse 1363 and H2L2 mouse 1359.

FIG. 4. Determination of binding affinities of individual monoclonal antibodies cloned and recombinantly expressed, as indicate, to hROR2-ECD as measured by surface plasmon resonance (SPR).

FIG. 5. Determination of species cross-reactivity of recombinant, monoclonal anti-hROR2 antibodies to mouse and cynomolgus ROR2 as evaluated by ELISA.

FIG. 6. (A) Chemical structures of pentaglycine-modified PNU derivative (“G5-PNU”), (B) triglycine-modified PNU derivative (“G3-PNU”).

FIG. 7. FACS analysis for hROR2 expression of mouse breast cancer cell line EMT-6 engineered to stably overexpress hROR2.

FIG. 8. Evaluation of in vitro cell killing activity of antibody drug conjugates comprising the PNU toxin (G5-PNU) site-specifically conjugated to the IgH and IgL chains of selected anti-hROR2 antibodies, as indicated, using EMT-6 cells engineered to stably express hROR2 according to Example 8.

FIG. 9. Evaluation of in vitro cell killing activity of antibody drug conjugates comprising novel hROR2 monoclonal antibody clone MK-3B12 conjugated to either the PNU toxins G3-PNU or G5-PNU using EMT-6 cells engineered to stably express hROR2 according to Example 9. An isotype-matched control ADC comprising the CD30-specific antibody clone brentuximab (clone Ac10) and site-specifically conjugated to G5-PNU, as well as CD30 expressing Karpas-299 cells were used for control experiments, as indicated.

FIG. 10. Evaluation of in vitro cell killing activity of antibody drug conjugates comprising the PNU toxin (G5-PNU) site-specifically conjugated to the IgH and IgL chains of selected anti-hROR2 antibodies, as indicated, using EMT-6 cells engineered to stably express hROR2 according to Example 10.

FIG. 11. Determination of species cross-reactivity of recombinant, monoclonal anti-hROR2 antibodies to mouse and cynomolgus ROR2 as evaluated by ELISA.

DETAILED DESCRIPTION OF THE INVENTION

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5′→3′.

In one aspect, the invention provides novel, high-affinity binding domains of fully human monoclonal antibodies that specifically bind to the extracellular domain of receptor tyrosine kinase-like orphan receptor 2 (ROR2). In one embodiment, ROR2 is human ROR2 (hROR2).

Such monoclonal antibodies have been selected from diverse antibody libraries generated by challenging human antibody light and heavy variable domain transgenic mice (WO 2010/070263 A1, H2L2 mice) with recombinant extracellular domain of the hROR2 antigen followed by generating and screening cellular IgG1 display libraries from hROR2 challenged H2L2 mice using DNA-transposable vectors for transfection into 63-12 mouse preB-cells according to WO 2014/013026 A1. Mammalian cells displaying the antibodies according to WO 2014/013026 A1 were screened using antigen-specific cell sorting (FACS), including single-cell sorting. Eventually, antibodies secreted in the supernatant of single-cell sorted clones were subjected to antigen-binding evaluation by ELISA and secondary cancer cell killing assays (i.e., wherein a secondary antibody coupled to a toxin binds to the antigen-bound anti-hROR2 antibody at the cell surface, and is internalised to effect cell killing). Coding sequences of variable heavy and light chain domains have been identified from selected cell clones displaying hROR2-specific binding and functional characterization involving cell killing of hROR2 expressing target cells via a Fab-based toxin-conjugated secondary reagent. By this strategy novel fully human monoclonal antibodies for hROR2 of high quality and favorable functional properties have been identified.

In a second aspect of the invention, antibody drug conjugates (ADCs) based on said anti-ROR2 antibodies, antibody-based binding proteins or antigen-binding fragments thereof, with one or more toxins, and in particular with an ultra-potent anthracycline toxin, are provided. In particular, such ADCs are generated by site-specific conjugation, achieved by enzymatic conjugation using sortase enzyme as disclosed in WO 2014/140317 A1, which is incorporated by reference herein. One particular ultra-potent anthracycline toxin with high potency has been disclosed in WO 2016/102679 A1, which is incorporated by reference herein.

In a third aspect of the invention, antibody effector conjugates (AECs) based on said anti-ROR2 antibodies, antibody-based binding proteins or antigen-binding fragments thereof, conjugated to one or more labels, are provided.

Additionally, the invention provides chimeric antigen receptors (CARs) and T cells engineered with these CARs, i.e. CAR-T cells, employing said anti-ROR2 antibodies, derivatives, modified formats or fragments.

Additionally, the invention provides bi- or multispecific antibodies comprising the binding domains of the disclosed anti-ROR2 antibodies, in bi- or multispecific antibody formats comprising at least one binding domain specific for another target, for instance, but not limited to targets that recruit and/or activate cells of the immune system, like T cells or NK cells. Such other binding domains may be specific for CD3, CD16, CD32, CD56, CD64 or other markers specific for T and NK cells.

In another aspect, the present invention refers to isolated or substantially purified polynucleotides encoding the variable region of the immunoglobulin heavy chain or immunoglobulin light chain of the fully human anti-ROR2 antibody, antibody-based binding protein or antigen-binding fragment thereof, and to expression vectors harboring such polynucleotides, as well as to host cells transformed or transfected with these polynucleotides, and methods of making anti-ROR2 antibodies, antibody-based binding proteins and antigen-binding fragments.

In another aspect, the present invention refers to a fully human anti-ROR2 antibody, antibody-based binding protein or antigen-binding fragment thereof, or ADC, a bi- or multispecific antibody, or a chimeric antigen receptor (CAR), as described herein, for use in the treatment of a subject that is suffering from, at risk of developing, and/or diagnosed with a neoplastic disease.

In another aspect, the present invention refers to a method for treating a subject suffering from, at risk of developing, and/or diagnosed with a neoplastic disease with a human anti-hROR2 antibody, antibody-based binding protein or antigen-binding fragment thereof, or ADC, or a bi- or multispecific antibody, or a CAR engineered cell, as described herein.

In another aspect, the present invention refers to a method for detecting a neoplastic disease or an immune disease or disorder suitable for treatment with an anti-ROR2 antibody, antibody-based binding protein or antigen-binding fragment thereof, or AEC.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. In addition, the following definitions are provided to assist the reader in the practice of the invention.

The term “human antibody” refers to an antibody, antibody-based binding protein, modified antibody format retaining target binding capacity, or antibody derivative or fragment retaining target binding capacity that contains sequences derived from human immunoglobulins such that substantially all of the heavy and light chain CDR1 and CDR2 regions are of human origin, and substantially all of the heavy and light chain FR regions 1, 2, 3, and 4 correspond to those of a human immunoglobulin sequence either with or without a limited number of somatic mutations that may be introduced into individual heavy and light chain CDR1 and CDR2 and FR1, 2, 3, and 4 variable domain sequences.

The terms “antibody”, “antibody-based binding protein”, “modified antibody format retaining target binding capacity”, “antibody derivative or fragment retaining target binding capacity” refer to polypeptide chain(s) which exhibit a strong monovalent, bivalent or polyvalent binding to a given antigen, epitope or epitopes. Antibodies, antibody-based binding proteins and antigen-binding fragments used in the invention can be generated using any suitable technology, e.g., hybridoma technology, ribosome display, phage display, gene shuffling libraries, semi-synthetic or fully synthetic libraries or combinations thereof. Antibodies, antibody-based binding proteins and antigen-binding fragments of the invention include intact antibodies and antibody fragments or antigen-binding fragments that contain the antigen-binding portions of an intact antibody and retain the capacity to bind the cognate antigen. Unless otherwise specified herein, all peptide sequences, including all antibody and antigen-binding fragment sequences are referred to in N→C order.

An intact antibody typically comprises at least two heavy (H) chains (about 50-70 kD) and two light (L) chains (about 25 kD) inter-connected by disulfide bonds. The recognized immunoglobulin genes encoding antibody chains include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Each heavy chain of an antibody is comprised of a heavy chain variable region (VH) and a heavy chain constant region. In the case of IgG, the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system and the first component (Clq) of the classical complement system. Monoclonal antibodies (mAbs) consist of identical antibodies molecules.

The VH and VL regions of an antibody can be further subdivided into regions of hypervariability, also termed complementarity-determining regions (CDRs), which are interspersed with the more conserved framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The locations of CDR and FR regions and a numbering system have been defined, e.g., the IMGT system (Lefranc M P et al., 2015), or the Kabat numbering scheme.

Antibodies, antibody-based binding proteins and antigen-binding fragments of the invention also encompass single chain antibodies. The term “single chain antibody” refers to a polypeptide comprising a VH domain and a VL domain in polypeptide linkage, generally linked via a spacer peptide, and which may comprise additional domains or amino acid sequences at the amino- and/or carboxyl-termini. For example, a single-chain antibody may comprise a tether segment for linking to the encoding polynucleotide. As an example, a single chain variable region fragment (scFv) is a single-chain antibody. Compared to the VL and VH domains of the Fv fragment that are coded for by separate genes, a scFv has the two domains joined (e.g., via recombinant methods) by a synthetic linker. This enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules.

Examples of antibody-based binding proteins are polypeptides in which the binding domains of the antibodies are combined with other polypeptides or polypeptide domains, e.g. alternative molecular scaffolds, Fc-regions, other functional or binding domains of other polypeptides or antibodies resulting in molecules with addition binding properties, e.g. bi- or multispecific proteins or antibodies. Such polypeptides can create an arrangement of binding or functional domains normally not found in naturally occurring antibodies or antibody fragments.

Examples of antigen-binding fragments include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an intact antibody; (v) disulfide stabilized Fvs (dsFvs) which have an interchain disulfide bond engineered between structurally conserved framework regions; (vi) a single domain antibody (dAb) which consists of a VH or VL domain (see, e.g., Ward et al., Nature 341:544-546, 1989); and (vii) an isolated complementarity determining region (CDR) as a linear or cyclic peptide.

The anti-ROR2 antibodies, antibody-based binding proteins and antigen-binding fragments described herein can be produced by enzymatic or chemical modification of the intact antibodies, or synthesized de novo using recombinant DNA methodologies. Methods for generating these antibodies, antibody-based binding proteins and antigen-binding molecules are all well known in the art. In particular, scFv antibodies can be obtained using methods described in, e.g., Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988. Fv antibody fragments can be generated as described in Skerra and PlUckthun, Science 240:1038-41, 1988. Disulfide-stabilized Fv fragments (dsFvs) can be made using methods described in, e.g., Reiter et al., Int. J. Cancer 67:113-23, 1996. Similarly, single domain antibodies (dAbs) can be produced by a variety of methods described in, e.g., Ward et al., Nature 341:544-546, 1989; and Cai and Garen, Proc. Natl. Acad. Sci. USA 93:6280-85, 1996. Camelid single domain antibodies can be produced using methods well known in the art, e.g., Dumoulin et al., Nat. Struct. Biol. 11:500-515, 2002; Ghahroudi et al., FEBS Letters 414:521-526, 1997; and Bond et al., J. Mol. Biol. 332:643-55, 2003. Other types of antigen-binding fragments (e.g., Fab, F(ab′)2 or Fd fragments) can also be readily produced with routinely practiced immunology methods. See, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998.

The anti-ROR2 antibodies, antibody-based binding proteins or antigen-binding fragments of the invention can be produced by any suitable technique, for example, using any suitable eukaryotic or non-eukaryotic expression system. In certain embodiments, the antibody, antibody-based binding protein or antigen-binding fragment is produced using a mammalian expression system. Some specific techniques for generating the antibodies, antibody-based binding proteins or antigen-binding fragments or antigen-binding fragments of the invention are exemplified herein. In some embodiments, the antibodies, antibody-based binding proteins or antigen-binding fragments of the invention can be produced using a suitable non-eukaryotic expression system such as a bacterial expression system. Bacterial expression systems can be used to produce fragments such as a F(ab)2, Fv, scFv, IgGACH2, F(ab′)2, scFv2CH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc, (scFv)2, and diabodies. Techniques for altering DNA coding sequences to produce such fragments are known in the art.

The term “conservatively modified variant” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein.

For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

For polypeptide sequences, “conservatively modified variants” refer to a variant which has conservative amino acid substitutions, amino acid residues replaced with other amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c, 1970; by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; by the search for similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis.); or by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou ed., 2003)). Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively.

Percent (%) identity of peptide sequences can be calculated, for example, as 100×[(identical positions)/min(TGA, TGB)], where TGA and TGB are the sum of the number of residues and internal gap positions in peptide sequences A and B in the alignment that minimizes TGA and TGB. See, e.g., Russell et al, J. Mol. Biol., 244: 332-350 (1994).

Artificial T cell receptors (also known as chimeric T cell receptors, chimeric immunoreceptors, chimeric antigen receptors (CARs) or T-bodies) are engineered receptors, which graft an arbitrary specificity onto an immune effector cell. Typically, these receptors are used to graft the specificity of a monoclonal antibody onto a T cell; with transfer of their coding sequence facilitated by retroviral or lentiviral vectors or by transposons. CAR-engineered T cells are genetically engineered T cells armed with chimeric receptors whose extracellular recognition unit is comprised of an antibody-derived recognition domain and whose intracellular region is derived from lymphocyte stimulating moiety(ies). The structure of the prototypic CAR is modular, designed to accommodate various functional domains and thereby to enable choice of specificity and controlled activation of T cells. The preferred antibody-derived recognition unit is a single chain variable fragment (scFv) that combines the specificity and binding residues of both the heavy and light chain variable regions of a monoclonal antibody, but also other antibody-derived formats like Fab fragments, V-domains etc. may be employed to confer a desired CAR specificity to CAR engineered T cells. The most common lymphocyte activation moieties include a T-cell costimulatory (e.g. CD28) domain in tandem with a T-cell triggering (e.g. CD3zeta) moiety, but also other signaling domains, like 4-1BB can be employed in the intracellular portion of CARs. By arming effector lymphocytes (such as T cells and natural killer cells) with such chimeric receptors, the engineered cell is redirected with a predefined specificity to any desired target antigen, in a non-HLA restricted manner. CAR constructs are introduced ex vivo into T cells from peripheral lymphocytes of a given patient using retroviral or lentiviral vectors or transposons. Following infusion of the resulting CAR-engineered T cells back into the patient, they traffic, reach their target site, and upon interaction with their target cell or tissue, they undergo activation and perform their predefined effector function. Therapeutic targets for the CAR approach include cancer and HIV-infected cells, or autoimmune effector cells.

The terms “treat,” “treating,” “treatment,” and “therapeutically effective” used herein do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment recognized by one of ordinary skill in the art as having a potential benefit or therapeutic effect. In this respect, the inventive method can provide any amount of any level of treatment. Furthermore, the treatment provided by the inventive method can include the treatment of one or more conditions or symptoms of the disease being treated.

A “vector” is a replicon, such as plasmid, phage or cosmid, to which another polynucleotide segment may be attached so as to bring about the replication of the attached segment. Vectors capable of directing the expression of genes encoding for one or more polypeptides are referred to as “expression vectors”.

DETAILED DESCRIPTION OF EMBODIMENTS

Invention aspects relating to binding of human ROR2

In one aspect, the present invention refers to a human anti-ROR2 antibodies, antibody-based binding proteins (incl. bi- or multi-specific antibodies), antigen-binding fragments thereof, AECs, ADCs, or CARs having the same binding specificity for ROR2 (and especially for hROR2 comprising or consisting of SEQ ID NO. 1), i.e., binding to the same ROR2 epitope and/or competing for ROR2 binding, as ROR2 specific antibodies containing an immunoglobulin heavy chain variable region sequence and an immunoglobulin light chain variable region sequence pair, as per Table 2. In particular, the present invention refers to human anti-ROR2 antibodies, antibody-based binding proteins (incl. bi- or multi-specific antibodies), antigen-binding fragments thereof, AECs, ADCs, or CARs having the same binding specificity for ROR2 (and especially for hROR2 comprising or consisting of SEQ ID NO. 1).

In one aspect, a fully human antibody, or a derivative, modified format or fragment thereof, is provided, which specifically binds to the extracellular domain of receptor tyrosine kinase-like orphan receptor 2 (ROR2).

According to one embodiment, the antibody, derivative, modified format or fragment is an antibody, an antibody-based binding protein, a modified antibody format retaining target binding capacity, antibody derivative or fragment retaining target binding capacity.

In one embodiment, the antibody, derivative, modified format or fragment, (i) binds to the same ROR2 epitope as and/or (ii) competes for ROR2 binding with an antibody comprising an Ig heavy chain variable region sequence and an Ig light chain variable region sequence, respectively, shown in:

(i) SEQ ID NO:2 and SEQ ID NO:3;

(ii) SEQ ID NO:4 and SEQ ID NO:5;

(iii) SEQ ID NO:6 and SEQ ID NO:7;

(iv) SEQ ID NO:8 and SEQ ID NO:9;

(v) SEQ ID NO:10 and SEQ ID NO:11;

(vi) SEQ ID NO:12 and SEQ ID NO:13;

(vii) SEQ ID NO:14 and SEQ ID NO:15;

(vii) SEQ ID NO:16 and SEQ ID NO:17;

(ix) SEQ ID NO:18 and SEQ ID NO:19;

(x) SEQ ID NO:20 and SEQ ID NO:21;

(xi) SEQ ID NO:22 and SEQ ID NO:23; or

(xii) SEQ ID NO:24 and SEQ ID NO:25.

In one other embodiment, the antibody, derivative, modified format or fragment comprises a heavy chain variable region sequence and a light chain variable region sequence, respectively, one or both of which are at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99% and most preferably 100% identical, to the heavy chain variable region sequence/light chain variable region sequence pairs shown above.

TABLE 1 SEQ ID NO. 1: amino acid sequence from the extracellular domain of human ROR2 (hROR2), based on sequence NP_004551.2 from GenBank SEQ ID NO. Amino Acid Sequence SEQ ID NO. 1 EVEVLDPNDPLGPLDGQDGPIPTLKGYFLNFLEPVNNITIVQGQTAILHCKVAGNPPPNV RWLKNDAPVVQEPRRIIIRKTEYGSRLRIQDLDTTDTGYYQCVATNGMKTITATGVLFVR LGPTHSPNHNFQDDYHEDGFCQPYRGIACARFIGNRTIYVDSLQMQGEIENRITAAFTMI GTSTHLSDQCSQFAIPSFCHFVFPLCDARSRTPKPRELCRDECEVLESDLCRQEYTIARS NPLILMRLQLPKCEALPMPESPDAANCMRIGIPAERLGRYHQCYNGSGMDYRGTASTTKS GHQCQPWALQHPHSHHLSSTDFPELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPS CSPRDSSKMG

TABLE 2 Amino acid sequences of SEQ ID NO. 2-25 and 75-92, comprising variable and constant domains, wherein the constant domain is underlined SEQ ID NO. Name Amino Acid Sequence (with constant domain underlined) SEQ ID NO. 2 EVQLVESGPGLLKPSETLSLTCAVSGYSISSGYYWGWIRQPPGKGLEWIGSIYQSGSTHY MK-3B12 HC NPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCAREDRAGWYPFDCWGQGTLVTVSS amino acid ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS sequence GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGR SEQ ID NO. 3 DIVMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPS MK-3B12 LC RFSGSGSGTEFTLTISSLQPDDFATYYCQQYNNYWTFGQGTKVEIKRTVAAPSVFIFPPS amino acid DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL sequence SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 4 EVQLLETGGGVVQPGRSLRLSCVASGFTFRSHGMHWVRQAPGKGLEWVALIWYDGSKKYY MK-7C3 HC ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGAGLYLDYWGQGTLVTVSSAS amino acid TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL sequence YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 5 AIRMTQSPSTLSASVGDRVTITCRASQTISNWLAWFQQKPGKAPKVLIYKASSLESGVPS MK-7C3 LC RFSGSGSGTEFTLTISSLQPDDFASYYCQQYNSYSYTFGQGTRLEIKRTVAAPSVFIFPP amino acid SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT sequence LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 6 EVQLVESGGGVVQPGRSLRLSCAASGFTFRSYGMHWVRQAPGKGLEWVAIIWYDGSKKYY GK-1E5 HC TDSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPGIAMTGLDYWGQGTLVTVSSA amino acid STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG sequence LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 7 DIQLTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPS GK-1E5 LC RFSGSGSGTEFTLTISSLQPDDFATYYCQQYNNYWTFGQGTKVEIKRTVAAPSVFIFPPS amino acid DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL sequence SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 8 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWNDGSNKYY GK-5A1 HC ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGSGWYDYYYGMDVWGQGTTVT amino acid VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL sequence QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 9 EIVLTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPS GK-5A1 LC RFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYWTFGQGTKVDIKRTVAAPSVFIFPPS amino acid DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL sequence SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 10 QVQLQESGGGVVQPGRSLRLSCAASGFTFRSYGMHWVRQAPGKGLEWVAIIWYDGSKKYY GK-2G8 HC TDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPGVAMTGLDLWGQGTLVTVSSA amino acid STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG sequence LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 11 EIVMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPS GK-2G8 LC RFSGSGSGTEFTLTISSLQPDDFATYYCQQYNNYWTFGQGTKVDIKRTVAAPSVFIFPPS amino acid DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL sequence SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 12 QVTLKESGGDVVQPGRSLRLSCAASGFTFRTYGMHWVRQAPGKGPEWVALIWYDGSKKYY GK-5E1 HC ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRVRFGELYFQHWGQGTLVTVSSA amino acid STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG sequence LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 13 DIVMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPS GK-5E1 LC RFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYSFGQGTKLEIKRTVAAPSVFIFPP amino acid SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT sequence LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 14 QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVALIWYDGSNKYY GK-6B10 HC ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVAAALHFHYWGQGTLVTVSSAS amino acid TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL sequence YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 15 DIVMTQSPSTLSASVGDRVTITCRASQSIDNWLAWYQQKPGKAPKVLIYKASSLESGVPS GK-6B10 LC RFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYTFGQGTKLEIKRTVAAPSVFIFPP amino acid SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT sequence LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 16 QITLKESGGGVVQPGRSLRLSCAASGFTFRTYGMHWVRQAPGKGLEWVALIWYDGSNKYY GK-5G12 HC ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCIRVKFGDLYFQHWGQGTLVTVSSA amino acid STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG sequence LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 17 EIVLTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPS GK-5G12 LC RFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPYTFGQGTKLEIKRTVAAPSVFIFPP amino acid SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT sequence LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 18 QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAIIWYDGSNKYY GK-21D3 HC ADSVKGRFTISRDNSKNTLYLQMNSLRDEDTAVYYCARMGAINRGGGGFDYWGQGTLVTV amino acid SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ sequence SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 19 DIQLTQSPSSLSASIGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPS GK-21D3 LC RFSGSGSGTDFTLTISSLQPEDVSTYYCQKYNSAPWTFGQGTKVDIKRTVAAPSVFIFPP amino acid SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT sequence LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 20 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWFDGTNKHY MK-24C10 HC ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDKGEWFGELRYYYYGMDVWGQG amino acid TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF sequence PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 21 EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRA MK-24C10 LC SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTKLEIKRTVAAPSV amino acid FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL sequence SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 22 EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGDINHSRTTNYN MK-24F9 HC PSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGEQWLVPFDYWDQGTLVTVSSA amino acid STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG sequence LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 23 EIVMTQSPSTLSASVGDRVTITCRASQSISHWLAWYQQKPGKAPKLLIYKASSLKSGVPS MK-24F9 LC RFNGSGSGTEFTLTISSLQPDDFATYYCQHYNTYSRTFGQGTKVDIKRTVAAPSVFIFPP amino acid SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT sequence LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 24 EVQLVESGGGLVQSGGSLRLSCAASGFTFSSQRLSWVRQAPGKGLEWVANIKQDGSEKNY GK-22G12 HC VDSVRGRFTISRDIAKNSLYLQMNSLRAEDTAVYYCARDGYRNGWHIPEDYWGQGTLVTV amino acid SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ sequence SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 25 DIVMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPS GK-22G12 LC RFSGSGSGTDFTLTISSLQPEDVSTYYCQKHNRAPWTFGQGTKLEIKRTVAAPSVFIFPP amino acid SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT sequence LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 75 EVQLLESGGGVVQPGRSLRLSCAASGFTFRSYGMHWVRQAPGKGLEWVAIIWYDGSKKYY GK-1H2 HC TDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPGIAMTGLDYWGQGTLVTVSSA amino acid STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG sequence LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 76 EIVMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPS GK-1H2 LC RFSGSGSGTEFTLTISSLQPDDFATYYCQQYNNYWTFGQGTKLEIKRTVAAPSVFIFPPS amino acid DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL sequence SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 77 QVQLVQSGGGVVQPGRSLRLSCAASGFTFRSYGMHWVRQAPGKGLEWVAIIWYDGSKKYY GK-2A9 HC TDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPGVAMTGLDLWGQGTLVTVSSA amino acid STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG sequence LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 78 EIVLTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPS GK-2A9 LC RFSGSGSGTEFTLTISSLQPDDFATYYCQQYNNYWTFGQGTKVDIKRTVAAPSVFIFPPS amino acid DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL sequence SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 79 EVQLQESGGGVVQPGRSLRLSCAASGFTFRTYGMHWVRQAPGKGLEWVALIWYDGSNKYY GK-5A6 HC ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCIRVKFGDLYFQHWGQGTLVTVSSA amino acid STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG sequence LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 80 DVVMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPS GK-5A6 LC RFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYTFGQGTKLEIKRTVAAPSVFIFPP amino acid SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT sequence LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 81 QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAIIWYDGSNKYY GK-21F1 HC ADSVKGRFTISRDNSKNTLYLQMNSLRDEDTAVYYCARMGAINRGGGGFDYWGQGTLVTV amino acid SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ sequence SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 82 DIQLTQSPSSLSASIGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPS GK-21F1 LC RFSGSGSGTDFTLTISSLQPEDVSTYYCQKYNSAPWTFGQGTKVDIKRTVAAPSVFIFPP amino acid SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT sequence LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 83 EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWFDGTNKHY MK-24C12 HC ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDKGEWFGELRYYYYGMDVWGQG amino acid TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF sequence PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 84 EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRA MK-24C12 LC SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTKLEIKRTVAAPSV amino acid FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL sequence SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 85 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGITNYN GK-21G5 HC PSLKSRLTVSVDTSKNQFSLKLSSVTAADTAVYYCARGGDQWLVPFDNWGQGTLVTVSSA amino acid STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG sequence LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 86 DIVMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPS GK-21G5 LC RFSGSGSGTDFTLTISSLQPEDVSTYYCQKHNRAPWTFGQGTKLEIKRTVAAPSVFIFPP amino acid SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT sequence LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 87 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGITNYN GK-23A8 HC PSLKSRLTVSVDTSKNQFSLKLSSVTAADTAVYYCARGGDQWLVPFDNWGQGTLVTVSSA amino acid STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG sequence LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 88 EIVMTQSPSTLSASVGDRVTITCRASQSISHWLAWYQQKPGKAPKLLIYKASSLKSGVPS GK-23A8 LC RFNGSGSGTEFTLTISSLQPDDFATYYCQHYNTYSRTFGQGTKVDIKRTVAAPSVFIFPP amino acid SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT sequence LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 89 RVQLVQSGGGLVQSGGSLRLSCAASGFTFSSQRLSWVRQAPGKGLEWVANIKQDGSEKNY GK-21E6 HC VDSVRGRFTISRDIAKNSLYLQMNSLRAEDTAVYYCARDGYRNGWHIPEDYWGQGTLVTV amino acid SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ sequence SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 90 DVVMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPS GK-21E6 LC RFSGSGSGTDFTLTISSLQPEDVSTYYCQKHNRAPWTFGQGTKLEIKRTVAAPSVFIFPP amino acid SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT sequence LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 91 EVQLLESGGGLVQSGGSLRLSCAASGFTFSSQRLSWVRQAPGKGLEWVANIKQDGSEKNY GK-22E12 HC VDSVRGRFTISRDIAKNSLYLQMNSLRAEDTAVYYCARDGYRNGWHIPEDYWGQGTLVTV amino acid SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ sequence SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 92 AIRMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPS GK-22E12 LC RFSGSGSGTDFTLTISSLQPEDVSTYYCQKHNRAPWTFGQGTKVEIKRTVAAPSVFIFPP amino acid SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT sequence LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Assays for assessing binding competition include, but are not limited to, radioactive material labeled immunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), sandwich ELISA assays, fluorescence activated cell sorting (FACS) assays and Biacore (SPR) assays. In conducting an antibody competition assay between a control antibody and a test antibody, one may first label the reference with a detectable label, such as a fluorophore, biotin or an enzymatic (or even radioactive) label to enable subsequent identification. If the test antibody competes with the labeled control antibody, the intensity will be decreased relative to a control reaction carried out without test antibody.

Methods for determining binding epitope include, but are not limited to, assessment of binding to an array of oligopeptides having (overlapping) amino acid sequences from the ROR2 sequence, resolution of a crystal or NMR structure of the antibody, antibody-based binding protein or antigen-binding fragment thereof with ROR2, by assessment of binding loss of antibodies, antibody-based binding proteins, antigen-binding fragments thereof to ROR2 comprising one or more amino acid mutations (“high throughput mutagenesis”) and by hydrogen-deuterium exchange to assess the solvent accessible surface of the ROR2 complex with the antibody, antibody-based binding protein or antigen-binding fragment (Abbott M. et al, 2014).

In a preferred embodiment, fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, AECs, ADCs, or CARs having the same binding specificity for ROR2 (and especially for hROR2 comprising or consisting of SEQ ID NO. 1), i.e., binding to the same ROR2 epitope and/or competing for ROR2 binding, as ROR2 specific antibodies containing an immunoglobulin heavy chain variable region (Ig HCVR) sequence and an immunoglobulin light chain variable region (Ig LCVR) sequence, respectively, shown in: (i) SEQ ID NO:2 and SEQ ID NO:3; (ii) SEQ ID NO:6 and SEQ ID NO:7; (iii) SEQ ID NO:10 and SEQ ID NO:11; (iv) SEQ ID NO:12 and SEQ ID NO:13; (v) SEQ ID NO:14 and SEQ ID NO:15; (vi) SEQ ID NO:16 and SEQ ID NO:17; (vii) SEQ ID NO:4 and SEQ ID NO:5; (viii) SEQ ID NO:8 and SEQ ID NO:9; (ix) SEQ ID NO:18 and SEQ ID NO:19; (x) SEQ ID NO:24 and SEQ ID NO:25; (xi) SEQ ID NO:20 and SEQ ID NO:21; and (xii) SEQ ID NO:22 and SEQ ID NO:23, and more preferably as shown in (i) SEQ ID NO:2 and SEQ ID NO:3; (ii) SEQ ID NO:8 and SEQ ID NO:9; and (iii) SEQ ID NO:20 and SEQ ID NO:21.

Type Ig HCVR Ig LCVR MK-3B12 SEQ ID NO: 2 SEQ ID NO: 3 GK-1E5 SEQ ID NO: 6 SEQ ID NO: 7 GK-2G8 SEQ ID NO: 10 SEQ ID NO: 11 GK-5E1 SEQ ID NO: 12 SEQ ID NO: 13 GK-6B10 SEQ ID NO: 14 SEQ ID NO: 15 GK-5G12 SEQ ID NO: 16 SEQ ID NO: 17 MK-7C3 SEQ ID NO: 4 SEQ ID NO: 5 GK-5A1 SEQ ID NO: 8 SEQ ID NO: 9 MK-21D3 SEQ ID NO: 18 SEQ ID NO: 19 GK-22G12 SEQ ID NO: 24 SEQ ID NO: 25 MK-24C10 SEQ ID NO: 20 SEQ ID NO: 21 MK-24F9 SEQ ID NO: 22 SEQ ID NO: 23

In various embodiments, an antibody, antibody-based binding protein, antigen-binding fragment thereof, AEC, ADC or CAR is considered to compete with a control antibody if it decreases binding of the control antibody by at least about 20% or more, for example, by at least about 20%, 30%, 40%, 50%, 60%. 70%, 80%, 90%, 95% or even more, or by a percentage ranging between any of the foregoing values, at a control antibody concentration that is 80% of maximal binding under the specific assay conditions used, and a test antibody or antigen-binding fragment concentration that is 10-fold higher than the control antibody concentration.

Preferred embodiments relative to the foregoing invention aspects

In any of the foregoing aspects of the invention, the fully human anti-ROR2 antibody, antibody-based binding protein, antigen-binding fragment thereof, AEC, ADC or CAR preferably binds to ROR2 comprising or consisting of SEQ ID NO. 1 with a K_(d) of less than 100 nM, more preferably of less than 75 nM, even more preferably of less than 50 nM and most preferably of less than 25 nM.

In any of the foregoing aspects of the invention, the fully human anti-ROR2 antibody, antibody-based binding protein, antigen-binding fragment thereof, AEC, ADC or CAR preferably binds to ROR2 comprising or consisting of SEQ ID NO. 1 with a k_(on) of greater than 1×10⁴ M⁻¹s⁻¹, more preferably of greater than 1×10⁵ M⁻¹s⁻¹, even more preferably of greater than 1×10⁶ s⁻¹.

In any of the foregoing aspects of the invention, the fully human anti-ROR2 antibody, antibody-based binding protein, antigen-binding fragment thereof, AEC, ADC or CAR preferably binds to ROR2 comprising or consisting of SEQ ID NO. 1 with a k_(off) of less than 1×10⁻² s⁻¹, more preferably of less than 1×10⁻³ s⁻¹, and even more preferably of less than 1×10⁻⁴ s⁻¹.

Binding affinity is generally expressed in terms of equilibrium association or dissociation constants (K_(a) or K_(d), respectively), which are in turn reciprocal ratios of dissociation and association rate constants (k_(off) and k_(on), respectively). Thus, equivalent affinities may correspond to different rate constants, so long as the ratio of the rate constants remains the same. Binding affinities and/or rate constants can be determined using techniques well known in the art or described herein, such as, for example, ELISA, isothermal titration calorimetry (ITC), Biacore (SPR), biolayer inferometry or fluorescent polarization.

The invention also relates to fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs comprising a V_(H) chain comprising three CDRs wherein:

-   -   V_(H) CDR no. 1 comprises or consists of peptide sequence         GYSISSGYY (SEQ ID NO. 26) or GX₁X₂FX₃X₄X₅X₆ (SEQ ID NO. 67)         where X₁=F or G, X₂=T or S, X₃=R or S, X₄=S, T, R or G, X₅=H, Y         or Q, X₆=G, Y or R; and/or     -   V_(H) CDR no. 2 comprises or consists of peptide sequence         IYQSGST (SEQ ID NO. 27), INHSRTT (SEQ ID NO. 59), INHSGIT (SEQ         ID NO. 93), or IX₇X₈DGX₉X₁₀K (SEQ ID NO. 68), where X₇=W or K,         X₈=Y, N, F or Q, X₉₌₅ or T, X₁₀=K, N or E; and/or     -   V_(H) CDR no. 3 comprises or consists of a peptide sequence         selected from:

(SEQ ID NO. 28) CAREDRAGWYPFDCW, (SEQ ID NO. 33) CARVGAGLYLDYW, (SEQ ID NO. 40) CAREGSGWYDYYYGMDVW, (SEQ ID NO. 62) CQHYNTYSRTF, (SEQ ID NO. 37) CARPGIAMTGLDYW, (SEQ ID NO. 95) CARGGDQWLVPFDNW, (SEQ ID NO. 47) CARVAAALHFHYW, (SEQ ID NO. 60) CARGGEQWLVPFDYW, (SEQ ID NO. 43) CARPGVAMTGLDLW, (SEQ ID NO. 49) CIRVKFGDLYFQHW, (SEQ ID NO. 44) CVRVRFGELYFQHW, (SEQ ID NO. 65) CARDGYRNGWHIPEDYW, (SEQ ID NO. 52) CARMGAINRGGGGFDYW, and (SEQ ID NO. 55) CARDKGEWFGELRYYYGMDVW.

The invention also relates to fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs comprising a V_(H) chain comprising three CDRs wherein:

-   -   V_(H) CDR no. 1 comprises or consists of peptide sequence         GYSISSGYY (SEQ ID NO. 26) or GX₁X₂FX₃X₄X₅X₆ (SEQ ID NO. 67)         where X₁=F or G, X₂=T or S, X₃=R or S, X₄=S, T, R or G, X₅=H, Y         or Q, X₆=G, Y or R; and     -   V_(H) CDR no. 2 comprises or consists of peptide sequence         IYQSGST (SEQ ID NO. 27), INHSRTT (SEQ ID NO. 59), or         IX₇X₈DGX₉X₁₀K (SEQ ID NO. 68), where X₇=W or K, X₈=Y, N, F or Q,         X₉=S or T, X₁₀=K, N or E; and     -   V_(H) CDR no. 3 comprises or consists of a peptide sequence         selected from:

(SEQ ID NO. 28) CAREDRAGWYPFDCW, (SEQ ID NO. 33) CARVGAGLYLDYW, (SEQ ID NO. 40) CAREGSGWYDYYYGMDVW, (SEQ ID NO. 62) CQHYNTYSRTF, (SEQ ID NO. 37) CARPGIAMTGLDYW, (SEQ ID NO. 95) CARGGDQWLVPFDNW, (SEQ ID NO. 47) CARVAAALHFHYW, (SEQ ID NO. 60) CARGGEQWLVPFDYW, (SEQ ID NO. 43) CARPGVAMTGLDLW, (SEQ ID NO. 49) CIRVKFGDLYFQHW, (SEQ ID NO. 44) CVRVRFGELYFQHW, (SEQ ID NO. 65) CARDGYRNGWHIPEDYW, (SEQ ID NO. 52) CARMGAINRGGGGFDYW, and (SEQ ID NO. 55) CARDKGEWFGELRYYYGMDVW.

The invention also relates to fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs comprising a V_(H) chain comprising three CDRs wherein:

-   -   V_(H) CDR no. 1 comprises or consists of peptide sequence         GYSISSGYY (SEQ ID NO. 26), GFTFRSHG (SEQ ID NO. 31), GGSFSGYY         (SEQ ID NO. 58), GFTFSSQR (SEQ ID NO. 63), or GFTFX₃X₄YG (SEQ ID         NO. 71) where X₃=R or S, and X₄=S, T or R, and preferably         comprises or consists of peptide sequence GYSISSGYY (SEQ ID NO.         26), GFTFRSYG (SEQ ID NO 36), GFTFRTYG (SEQ ID NO 94), GFTFSRYG         (SEQ ID NO 45), GFTFRSHG (SEQ ID NO. 31), GFTFSSYG (SEQ ID NO.         38), GGSFSGYY (SEQ ID NO. 58) or GFTFSSQR (SEQ ID NO. 63), and         more preferably comprises or consists of peptide sequence         GYSISSGYY (SEQ ID NO. 26) or GFTFSSYG (SEQ ID NO. 38); and     -   V_(H) CDR no. 2 comprises or consists of peptide sequence         IYQSGST (SEQ ID NO. 27), IWYDGSKK (SEQ ID NO 32), IWYDGSNK (SEQ         ID NO 46), IWNDGSNK (SEQ ID NO. 39), IWFDGTNK (SEQ ID NO. 54),         INHSRTT (SEQ ID NO. 59) or IKQDGSEK (SEQ ID NO. 64), and         preferably comprises or consists of peptide sequence IYQSGST         (SEQ ID NO. 27), IWNDGSNK (SEQ ID NO. 39), or IWFDGTNK (SEQ ID         NO. 54); and     -   V_(H) CDR no. 3 comprises or consists of a peptide sequence         selected from: CAREDRAGWYPFDCW (SEQ ID NO. 28), CARPGIAMTGLDYW,         CARPGVAMTGLDLW (DEQ ID NO. 43), CVRVRFGELYFQHW (SEQ ID NO. 44),         CARVAAALHFHYW (SEQ ID NO. 47), CIRVKFGDLYFQHW (SEQ ID NO. 49),         CARVGAGLYLDYW (SEQ ID NO. 33), CARPGIAMTGLDYW (SEQ ID NO. 37),         CAREGSGWYDYYYGMDVW (SEQ ID NO. 40), CARDKGEWFGELRYYYYGMDVW (SEQ         ID NO. 55), CARGGEQWLVPFDYW (SEQ ID NO. 60) or CARDGYRNGWHIPEDYW         (SEQ ID NO. 65), and preferably comprises or consists of a         peptide sequence selected from: CAREDRAGWYPFDCW (SEQ ID NO. 28),         CAREGSGWYDYYYGMDVW (SEQ ID NO. 40) or CARDKGEWFGELRYYYYGMDVW         (SEQ ID NO. 55).

In any of the foregoing aspect of the invention, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs comprising a V_(H) chain comprising three CDRs wherein:

-   -   V_(H) CDR no. 1 comprises or consists of a peptide sequence         selected from HC CDR1 sequences listed in Table 3; and/or     -   V_(H) CDR no. 2 comprises or consists of a peptide sequence         selected from HC CDR2 sequences listed in Table 3; and/or     -   V_(H) CDR no. 3 comprises or consists of a peptide sequence         selected from HC CDR3 sequences listed in Table 3.

In any of the foregoing aspect of the invention, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs comprising a V_(H) chain comprising three CDRs wherein:

-   -   V_(H) CDR no. 1 comprises or consists of a peptide sequence         selected from HC CDR1 sequences listed in Table 3; and     -   V_(H) CDR no. 2 comprises or consists of a peptide sequence         selected from HC CDR2 sequences listed in Table 3; and     -   V_(H) CDR no. 3 comprises or consists of a peptide sequence         selected from HC CDR3 sequences listed in Table 3.

In any of the foregoing aspect of the invention, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs comprising a V_(H) chain comprise three CDRs wherein V_(H) CDR no. 1, V_(H) CDR no. 2 and V_(H) CDR no. 3 comprises or consists of a peptide sequence according to a given HC CDR1, CDR2 and CDR3 triplet listed in Table 3.

In any of the foregoing aspect of the invention, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs comprising a V_(H) chain comprise three CDRs wherein V_(H) CDR no. 1, V_(H) CDR no. 2 and V_(H) CDR no. 3 comprises or consists of a peptide sequence according to the HC CDR1, CDR2 and CDR3 triplet of MK-3B12, GK-5A1, GK-21D3, MK-24C10, MK-24F9 or GK-22G12, and more preferably of MK-3B12, GK-5A1 or MK-24C10 listed in Table 3.

The invention further relates to anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs comprising a V_(L) chain comprising three CDRs wherein:

-   -   V_(L) CDR no. 1 comprises or consists of peptide sequence         QSLLHSNGYNY (SEQ ID NO. 56), QSIDNW (SEQ ID NO. 48), or         QX₁₁ISX₁₂X₁₃ (SEQ ID NO. 69) where X₁₁=S, T or G, X₁₂=S, N or H         and X₁₃=W or Y; and/or     -   V_(L) CDR no. 2 comprises or consists of peptide sequence KAS,         AAS or LGS; and/or     -   V_(L) CDR no. 3 comprises or consists of a peptide sequence         CX₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂F (SEQ ID NO. 70) where X₁₄=Q or M,         X₁₅=K, H or Q, X₁₆=H, Y or A, X₁₇=N or L, X₁₈=R, T, Q, S or N;         X₁₉=A, Y or T; X₂₀=P, S or W, X₂₁=W, R, Y or absent, X₂₂=S or T.

The invention further relates to anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs comprising a V_(L) chain comprising three CDRs wherein:

-   -   V_(L) CDR no. 1 comprises or consists of peptide sequence         QSLLHSNGYNY (SEQ ID NO. 56), QSIDNW (SEQ ID NO. 48), or         QX₁₁ISX₁₂X₁₃ (SEQ ID NO. 69) where X₁₁=S, T or G, X₁₂=S, N or H         and X₁₃=W or Y; and     -   V_(L) CDR no. 2 comprises or consists of peptide sequence KAS,         AAS or LGS; and     -   V_(L) CDR no. 3 comprises or consists of a peptide sequence         CX₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂F (SEQ ID NO. 70) where X₁₄=Q or M,         X₁₅=K, H or Q, X₁₆=H, Y or A, X₁₇=N or L, X₁₈=R, T, Q, S or N;         X₁₉=A, Y or T; X₂₀=P, S or W, X₂₁=W, R, Y or absent, X₂₂=S or T.

The invention further relates to anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs comprising a V_(L) chain comprising three CDRs wherein:

-   -   V_(L) CDR no. 1 comprises or consists of peptide sequence QSISSW         (SEQ ID NO. 29), QTISNW (SEQ ID NO. 34), QSIDNW (SEQ ID NO. 48),         QGISNY (SEQ ID NO. 50), QSLLHSNGYNY (SEQ ID NO. 56) or QSISHW         (SEQ ID NO. 61), and preferably comprises or consists of peptide         sequence QSISSW (SEQ ID NO. 29), or QSLLHSNGYNY (SEQ ID NO. 56);         and     -   V_(L) CDR no. 2 comprises or consists of peptide sequence KAS,         LGS or AAS, and preferably comprises or consists of peptide         sequence KAS or LGS; and     -   V_(L) CDR no. 3 comprises or consists of a peptide sequence         CQQYNNYWTF (SEQ ID NO. 30), CQQYNSYWTF (SEQ ID NO. 41),         CQQYNSYSYSF (SEQ ID NO. 42), CQQYNSYSYSF (SEQ ID NO. 44),         CQQYNSYSYTF (SEQ ID NO. 35), CQKYNSAPYTF (SEQ ID NO. 51),         CARMGAINRGGGGFDYW (SEQ ID NO. 52), CQKYNSAPWTF (SEQ ID NO. 53),         CMQALQTPYTF (SEQ ID NO. 57), CQHYNTYSRTF (SEQ ID NO. 62) or         CQKHNRAPWTF (SEQ ID NO. 66), and preferably comprises or         consists of a peptide sequence CQQYNNYWTF (SEQ ID NO. 30),         CQQYNSYWTF (SEQ ID NO. 41) or CMQALQTPYTF (SEQ ID NO. 57).

In any of the foregoing aspect of the invention, the fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs comprising a V_(L) chain comprising three CDRs wherein:

-   -   V_(L) CDR no. 1 comprises or consists of a peptide sequence         selected from LC CDR1 sequences listed in Table 3; and/or     -   V_(L) CDR no. 2 comprises or consists of a peptide sequence         selected from LC CDR2 sequences listed in Table 3; and/or     -   V_(L) CDR no. 3 comprises or consists of a peptide sequence         selected from LC CDR3 sequences listed in Table 3.

In any of the foregoing aspect of the invention, the fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs comprising a V_(L) chain comprising three CDRs wherein:

-   -   V_(L) CDR no. 1 comprises or consists of a peptide sequence         selected from LC CDR1 sequences listed in Table 3; and     -   V_(L) CDR no. 2 comprises or consists of a peptide sequence         selected from LC CDR2 sequences listed in Table 3; and     -   V_(L) CDR no. 3 comprises or consists of a peptide sequence         selected from LC CDR3 sequences listed in Table 3.

In any of the foregoing aspect of the invention, the fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs comprising a V_(L) chain comprise three CDRs wherein V_(L) CDR no. 1, V_(L) CDR no. 2 and V_(L) CDR no. 3 comprises or consists of a peptide sequence according to a given LC CDR1, CDR2 and CDR3 triplet listed in Table 3.

In any of the foregoing aspect of the invention, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs comprising a V_(L) chain comprise three CDRs wherein V_(L) CDR no. 1, V_(L) CDR no. 2 and

-   -   V_(L) CDR no. 3 comprises or consists of a peptide sequence         according to the LC CDR1, CDR2 and CDR3 triplet of MK-3B12,         GK-5A1, GK-21D3, MK-24C10, MK-24F9 or GK-22G12, and more         preferably of MK-3B12, GK-5A1 or MK-24C10 listed in Table 3.

In any of the foregoing aspect of the invention, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs comprise a V_(L) and V_(H) chain each comprising three CDRs comprising or consisting of, as per Table 3, the peptide sequence of the respective CDRs of: MK-3B12, MK-7C3, GK-1E5, GK-5E1, GK-2G8, GK-5A1, GK-6B10, GK-5G12, GK-21D3, MK-24C10, MK-24F9 or GK-22G12, and preferably of: MK-3B12, GK-5A1, GK-21D3, MK-24C10, MK-24F9 or GK-22G12, and more preferably of: MK-3B12, GK-5A1 or MK-24C10.

In any of the foregoing aspect of the invention, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs comprise a V_(L) and V_(H) chain each comprising three CDRs comprising or consisting of, as per Table 3, the peptide sequence of the respective CDRs of: MK-3B12, GK-1E5, GK-2G8, GK-5E1, GK-6B10 or GK-5G12, or of: GK-5A1, GK-21D3, MK-24C10, MK-24F9, MK-7C3 or GK-22G12, or more preferably of: MK-3B12, GK-5A1, GK-21D3, MK-24C10, MK-24F9 or GK-22G12, or even more preferably of: MK-3B12, GK-5A1 or MK-24C10.

In one embodiment, the antibody, antibody-based binding protein, modified antibody format retaining target binding capacity, antibody derivative or fragment retaining target binding capacity has cross-reactivity to (i) human ROR2 (hROR2) and (ii) at least one of cynomolgus ROR2 (cROR2) and murine RoR2 (mROR2).

As used herein, the term “cross-reactivity” means that the antibody, antibody-based binding protein, modified antibody format retaining target binding capacity, antibody derivative or fragment retaining target binding is capable to bind to (i) human ROR2 (hROR2) and (ii) at least one of cynomolgus ROR2 (cROR2) and murine RoR2 (mROR2), with sufficient affinity.

Said cross-reactivity has significant advantages in preclinical applications, because the same antibody that has been used for studies in mice and cynomolgous monkeys can later be used for clinical approval. Further, said cross-reactivity has significant advantages in diagnostic as well as scientific applications.

In one embodiment, the antibody, antibody-based binding protein, modified antibody format retaining target binding capacity, antibody derivative or fragment retaining target binding capacity comprises one of the following CDR sets:

-   -   a) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 38, 39 and         40, and light chain CDRs 1 and 3 as set forth in SEQ ID NOs 29         and 41, with light chain CDR 2 having the sequence KAS     -   b) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 26-28, and         light chain CDRs 1-3 as set forth in SEQ ID NOs 29, 30 and 73,     -   c) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 94, 32 and         44, and light chain CDRs 1 and 3 as set forth in SEQ ID NOs 29         and 42, with light chain CDR 2 having the sequence KAS     -   d) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 38, 46 and         52, and light chain CDRs 1 and 3 as set forth in SEQ ID NOs 50         and 53, with light chain CDR 2 having the sequence AAS     -   e) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 38, 54 and         55, and light chain CDRs 1 and 3 as set forth in SEQ ID NOs 56         and 57, with light chain CDR 2 having the sequence LGS,     -   f) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 58-60, and         light chain CDRs 1 and 3 as set forth in SEQ ID NOs 61, and 62,         with light chain CDR 2 having the sequence KAS,     -   g) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 63-65 with         light chain CDRs 1 and 3 as set forth in SEQ ID NOs 50 and 66,         with light chain CDR 2 having the sequence AAS,

The CDRs are comprised in a suitable protein framework so as to be capable to bind to hROR2 as well as to at least one of cynomolgus ROR2 (cROR2) and murine RoR2 (mROR2), with sufficient affinity.

In one embodiment, the antibody, antibody-based binding protein, modified antibody format retaining target binding capacity, antibody derivative or fragment retaining target binding capacity comprises one of the following sequence pairs:

-   -   a) the heavy chain variable region sequence of antibody GK-5A1         shown in SEQ ID NO. 8 and the light chain variable region         sequence of antibody GK-5A1 shown in SEQ ID NO. 9,     -   b) the heavy chain variable region sequence of antibody MK-3B12         shown in SEQ ID NO. 2 and the light chain variable region         sequence of antibody MK-3B12 shown in SEQ ID NO. 3,     -   c) the heavy chain variable region sequence of antibody GK-5E1         shown in SEQ ID NO. 12 and the light chain variable region         sequence of antibody GK-5E1 shown in SEQ ID NO. 13,     -   d) the heavy chain variable region sequence of antibody GK-21D3         shown in SEQ ID NO. 18 and the light chain variable region         sequence of antibody GK-5E1 shown in SEQ ID NO. 19,     -   e) the heavy chain variable region sequence of antibody MK-24C10         shown in SEQ ID NO. 20 and the light chain variable region         sequence of antibody MK-24C10 shown in SEQ ID NO. 21,     -   f) the heavy chain variable region sequence of antibody MK-24F9         shown in SEQ ID NO. 22 and the light chain variable region         sequence of antibody MK-24F9 shown in SEQ ID NO. 23,     -   g) the heavy chain variable region sequence of antibody GK-22G12         shown in SEQ ID NO. 24 and the light chain variable region         sequence of antibody GK-22G12 shown in SEQ ID NO. 25.

In one embodiment. the antibody, antibody-based binding protein, modified antibody format retaining target binding capacity, antibody derivative or fragment retaining target binding capacity bi- or multispecific, and comprises:

-   -   a first portion that binds to the extracellular domain of         receptor tyrosine kinase-like orphan receptor 2 (ROR2), and     -   at least a second portion that binds to an effector antigen         selected from the group consisting of CD3, CD16, NKG2D, NKp46,         CD2, CD28 and/or CD25.

Some sequences are shown in the following:

TABLE 3 SEQ ID NO. 26-66 and 93-95, CDR sequences Name Amino Acid Sequence of CDRs MK-3B12 HC CDR CDR1: GYSISSGYY (SEQ ID NO. 26) amino acid sequences CDR2: IYQSGST (SEQ ID NO. 27) CDR3: CAREDRAGWYPFDCW (SEQ ID NO. 28) MK-3B12 LC CDR CDR1: QSISSW (SEQ ID NO. 29) amino acid sequence CDR2: KAS CDR3: CQQYNNYWTF (SEQ ID NO. 30) MK-7C3 HC CDR CDR1: GFTFRSHG (SEQ ID NO. 31) amino acid sequence CDR2: IWYDGSKK (SEQ ID NO. 32) CDR3: CARVGAGLYLDYW (SEQ ID NO. 33) MK-7C3 LC CDR CDR1: QTISNW (SEQ ID NO. 34) amino acid sequence CDR2: KAS CDR3: CQQYNSYSYTF (SEQ ID NO. 35) GK-1E5 HC CDR CDR1: GFTFRSYG (SEQ ID NO. 36) amino acid sequence CDR2: IWYDGSKK (SEQ ID NO. 32) CDR3: CARPGIAMTGLDYW (SEQ ID NO. 37) GK-1E5 LC CDR CDR1: QSISSW (SEQ ID NO. 29) amino acid sequence CDR2: KAS CDR3: CQQYNNYWTF (SEQ ID NO. 30) GK-5A1 HC CDR CDR1: GFTFSSYG (SEQ ID NO. 38) amino acid sequence CDR2: IWNDGSNK (SEQ ID NO. 39) CDR3: CAREGSGWYDYYYGMDVW (SEQ ID NO. 40) GK-5A1 LC CDR CDR1: QSISSW (SEQ ID NO. 29) amino acid sequence CDR2: KAS CDR3: CQQYNSYWTF (SEQ ID NO. 41) GK-2G8 HC CDR CDR1: GFTFRSYG (SEQ ID NO. 36) amino acid sequence CDR2: IWYDGSKK (SEQ ID NO. 32) CDR3: CARPGVAMTGLDLW (SEQ ID NO. 43) GK-2G8 LC CDR CDR1: QSISSW (SEQ ID NO. 29) amino acid sequence CDR2: KAS CDR3: CQQYNNYWTF (SEQ ID NO. 30) GK-5E1 HC CDR CDR1: GFTFRTYG (SEQ ID NO. 94) amino acid sequence CDR2: IWYDGSKK (SEQ ID NO. 32) CDR3: CVRVRFGELYFQHW (SEQ ID NO. 44) GK-5E1 LC CDR CDR1: QSISSW (SEQ ID NO. 29) amino acid sequence CDR2: KAS CDR3: CQQYNSYSYSF (SEQ ID NO. 42) GK-6B10 HC CDR CDR1: GFTFSRYG (SEQ ID NO. 45) amino acid sequence CDR2: IWYDGSNK (SEQ ID NO. 46) CDR3: CARVAAALHFHYW (SEQ ID NO. 47) GK-6B10 LC CDR CDR1: QSIDNW (SEQ ID NO. 48) amino acid sequence CDR2: KAS CDR3: CQQYNSYSYTF (SEQ ID NO. 35) GK-5G12 HC CDR CDR1: GFTFRTYG (SEQ ID NO. 94) amino acid sequence CDR2: IWYDGSNK (SEQ ID NO. 46) CDR3: CIRVKFGDLYFQHW (SEQ ID NO. 49) GK-5G12 LC CDR CDR1: QGISNY (SEQ ID NO. 50) amino acid sequence CDR2: AAS CDR3: CQKYNSAPYTF (SEQ ID NO. 51) GK-21D3 HC CDR CDR1: GFTFSSYG (SEQ ID NO. 38) amino acid sequence CDR2: IWYDGSNK (SEQ ID NO. 46) CDR3: CARMGAINRGGGGFDYW (SEQ ID NO. 52) GK-21D3 LC CDR CDR1: QGISNY (SEQ ID NO. 50) amino acid sequence CDR2: AAS CDR3: CQKYNSAPWTF (SEQ ID NO. 53) MK-24C10 HC CDR CDR1: GFTFSSYG (SEQ ID NO. 38) amino acid sequence CDR2: IWFDGTNK (SEQ ID NO. 54) CDR3: CARDKGEWFGELRYYYYGMDVW (SEQ ID NO. 55) MK-24C10 LC CDR CDR1: QSLLHSNGYNY (SEQ ID NO. 56) amino acid sequence CDR2: LGS CDR3: CMQALQTPYTF (SEQ ID NO. 57) MK-24F9 HC CDR CDR1: GGSFSGYY (SEQ ID NO. 58) amino acid sequence CDR2: INHSRTT(SEQ ID NO. 59) CDR3: CARGGEQWLVPI,DYW (SEQ ID NO. 60) MK-24F9 LC CDR CDR1: QSISHW (SEQ ID NO. 61) amino acid sequence CDR2: KAS CDR3: CQHYNTYSRTF (SEQ ID NO. 62) GK-22G12 HC CDR CDR1: GFTFSSQR (SEQ ID NO. 63) amino acid sequence CDR2: IKQDGSEK (SEQ ID NO. 64) CDR3: CARDGYRNGWHIPEDYW (SEQ ID NO. 65) GK-22G12 LC CDR CDR1: QGISNY (SEQ ID NO. 50) amino acid sequence CDR2: AAS CDR3: CQKHNRAPWTF (SEQ ID NO. 66) GK-1H2 HC CDR CDR1: GFTFRSYG (SEQ ID NO. 36) amino acid sequence CDR2: IWYDGSKK (SEQ ID NO. 32) CDR3: CARPGIAMTGLDYW (SEQ ID NO. 37) GK-1H2 LC CDR CDR1: QSISSW (SEQ ID NO. 29) amino acid sequence CDR2: KAS CDR3: CQQYNNYWTF (SEQ ID NO. 30) GK-2A9 HC CDR CDR1: GFTFRSYG (SEQ ID NO. 36) amino acid sequence CDR2: IWYDGSKK (SEQ ID NO. 32) CDR3: CARPGVAMTGLDLW (SEQ ID NO. 43) GK-2A9 LC CDR CDR1: QSISSW (SEQ ID NO. 29) amino acid sequence CDR2: KAS CDR3: CQQYNNYWTF (SEQ ID NO. 30) GK-5A6 HC CDR CDR1: GFTFRTYG (SEQ ID NO. 94) amino acid sequence CDR2: IWYDGSNK (SEQ ID NO. 46) CDR3: CIRVKFGDLYFQHW (SEQ ID NO. 49) GK-5A6 LC CDR CDR1: QSISSW (SEQ ID NO. 29) amino acid sequence CDR2: KAS CDR3: CQQYNSYSYTF (SEQ ID NO. 35) GK-21F1 HC CDR CDR1: GFTFSSYG (SEQ ID NO. 38) amino acid sequence CDR2: IWYDGSNK (SEQ ID NO. 46) CDR3: CARMGAINRGGGGFDYW (SEQ ID NO. 52) GK-21F1 LC CDR CDR1: QGISNY (SEQ ID NO. 50) amino acid sequence CDR2: AAS CDR3: CQKYNSAPWTF (SEQ ID NO. 53) MK-24C12 HC CDR CDR1: GFTFSSYG (SEQ ID NO. 38) amino acid sequence CDR2: IWFDGTNK (SEQ ID NO. 54) CDR3: CARDKGEWFGELRYYYYGMDVW (SEQ ID NO. 55) MK-24C12 LC CDR CDR1: QSLLHSNGYNY (SEQ ID NO. 56) amino acid sequence CDR2: LGS CDR3: CMQALQTPYTF (SEQ ID NO. 57) GK-21G5 HC CDR CDR1: GGSFSGYY (SEQ ID NO. 58) amino acid sequence CDR2: INHSGIT(SEQ ID NO. 93) CDR3: CARGGDQWLVPFDNW (SEQ ID NO. 95) GK-21G5 LC CDR CDR1: QGISNY (SEQ ID NO. 50) amino acid sequence CDR2: AAS CDR3: CQKHNRAPWTF (SEQ ID NO. 66) GK-23A8 HC CDR CDR1: GGSFSGYY (SEQ ID NO. 58) amino acid sequence CDR2: INHSGIT(SEQ ID NO. 93) CDR3: CARGGDQWLVPFDNW (SEQ ID NO. 95) GK-23A8 LC CDR CDR1: QSISHW (SEQ ID NO. 61) amino acid sequence CDR2: KAS CDR3: CQHYNTYSRTF (SEQ ID NO. 62) GK-21E6 HC CDR CDR1: GFTFSSQR (SEQ ID NO. 63) amino acid sequence CDR2: IKQDGSEK (SEQ ID NO. 64) CDR3: CARDGYRNGWHIPEDYW (SEQ ID NO. 65) GK-21E6 LC CDR CDR1: QGISNY (SEQ ID NO. 50) amino acid sequence CDR2: AAS CDR3: CQKHNRAPWTF (SEQ ID NO. 66) GK-22E12 HC CDR CDR1: GFTFSSQR (SEQ ID NO. 63) amino acid sequence CDR2: IKQDGSEK (SEQ ID NO. 64) CDR3: CARDGYRNGWHIPEDYW (SEQ ID NO. 65) GK-22E12 LC CDR CDR1: QGISNY (SEQ ID NO. 50) amino acid sequence CDR2: AAS CDR3: CQKHNRAPWTF (SEQ ID NO. 66)

In all of the foregoing aspects of the invention, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs preferably comprises a heavy chain/light chain variable region pair, the sequences of which are substantially identical to the variable region of any pair of Table 2, and particularly to one selected from the following pairs (Table 2): (i) SEQ ID NO:2 and SEQ ID NO:3; (ii) SEQ ID NO:4 and SEQ ID NO:5; (iii) SEQ ID NO:6 and SEQ ID NO:7; (iv) SEQ ID NO:8 and SEQ ID NO:9; (v) SEQ ID NO:10 and SEQ ID NO:11; (vi) SEQ ID NO:12 and SEQ ID NO:13; (vii) SEQ ID NO:14 and SEQ ID NO:15; (vii) SEQ ID NO:16 and SEQ ID NO:17; (ix) SEQ ID NO:18 and SEQ ID NO:19; (x) SEQ ID NO:20 and SEQ ID NO:21; (xi) SEQ ID NO:22 and SEQ ID NO:23; or (xii) SEQ ID NO:24 and SEQ ID NO:25. In a preferred embodiment, the fully human anti-ROR2 antibody or antigen-binding fragment comprises a heavy chain/light chain variable region pair, the sequences of which are substantially identical to one selected from the following pairs (Table 2): (i) SEQ ID NO:2 and SEQ ID NO:3; (ii) SEQ ID NO:8 and SEQ ID NO:9; (iii) SEQ ID NO:18 and SEQ ID NO:19; (iv) SEQ ID NO:20 and SEQ ID NO:21; (v) SEQ ID NO:22 and SEQ ID NO:23; or (vi) SEQ ID NO:24 and SEQ ID NO:25. In a preferred embodiment, the fully human anti-ROR2 antibody or antigen-binding fragment comprises a heavy chain/light chain variable region pair, the sequences of which are substantially identical to one selected from the following pairs (Table 2): (i) SEQ ID NO:2; SEQ ID NO:3; (ii) SEQ ID NO:8 and SEQ ID NO:9 and (iii) SEQ ID NO:20 and SEQ ID NO:21.

In a preferred embodiment, “substantially identical” means at least 90%, preferably 95%, more preferably 100% identical to the respective the variable region amino acid sequence identified in the respective SEQ ID NO.

In some embodiments, the invention provides fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof that are conservatively modified variants of the human anti-ROR2 antibodies exemplified herein. Typically, the variable regions of these variants have an amino acid sequence that is identical to one of these exemplified sequences except for conservative substitutions at one or more amino acid residues.

In all of the foregoing aspects of the invention, the human antibodies, antibody-based binding proteins, antigen-binding fragments thereof is preferably an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, IgM, F(ab)2, Fv, scFv, IgGACH2, F(ab′)2, scFv2CH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc, (scFv)2, a non-depleting IgG, a diabody, or a bivalent antibody. In a more preferred embodiment, the human antibodies, antibody-based binding proteins, antigen-binding fragments thereof are an IgG1, IgG2, IgG3, IgG4, IgM.

In the case where the human antibodies, antibody-based binding proteins or antigen-binding fragments thereof (or ADCs or CARs made therewith) are bivalent, it is preferred that they bind to both human ROR2 and human CD3.

In a preferred embodiment, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs of the invention bind to human ROR2 and to cynomolgus ROR2 and compete for binding with an antibody comprising the variable domains of GK-5A1, MK-3B12, GK-5E1, MK-21D3, MK-24C10, MK-24F9 or MK-22G12, and preferably with an antibody comprising the variable domains of GK-5A1, MK-3B12, MK-24C10, MK-24F9 or MK-22G12.

In a preferred embodiment, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs of the invention bind to human and to cynomolgus ROR2. In a preferred embodiment, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs bind to cynomolgus ROR2 and to human ROR2 and comprise the CDRs of GK-5A1, MK-3B12, GK-5E1, MK-21D3, MK-24C10, MK-24F9 or MK-22G12, and preferably of GK-5A1, MK-3B12, MK-24C10, MK-24F9 or MK-22G12. In a preferred embodiment, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs of the invention bind to cynomolgus ROR2 and to human ROR2 and comprise the variable domains of GK-5A1, MK-3B12, GK-5E1, MK-21D3, MK-24C10, MK-24F9 or MK-22G12, and preferably of GK-5A1, MK-3B12, MK-24C10, MK-24F9. or MK-22G12

In a preferred embodiment, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs of the invention bind to human ROR2 and to mouse ROR2 and compete for binding with an antibody comprising the variable domains of GK-5A1, MK-24C10 or MK-24F9, and preferably with an antibody comprising the variable domains of GK-5A1.

In a preferred embodiment, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs of the invention bind to mouse ROR2 and to human ROR2. In a preferred embodiment, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs of the invention bind to mouse ROR2 and to human ROR2 and comprise the CDRs of GK-5A1, MK-24C10 or MK-24F9, and preferably of GK-5A1. In a preferred embodiment, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs of the invention bind to mouse ROR2 and to human ROR2 and comprise the variable domains of GK-5A1, MK-24C10 or MK-24F9, and preferably of GK-5A1.

The cross reactivity of the antibodies of the invention with mouse ROR2 is particularly surprising given that these antibodies originate from mouse.

In a preferred embodiment, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs of the invention bind to human ROR2, to cynomolgus ROR2 and to mouse ROR2 and compete for binding with an antibody comprising the variable domains of GK-5A1, MK-24C10 or MK-24F9, and preferably with an antibody comprising the variable domains of GK-5A1.

In a preferred embodiment, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs of the invention bind to cynomolgus ROR2, to mouse ROR2 and to human ROR2. In a preferred embodiment, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs of the invention bind to cynomolgus ROR2, to mouse ROR2 and to human and comprise the CDRs of GK-5A1, MK-24C10 or MK-24F9, and preferably of GK-5A1. In a preferred embodiment, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs of the invention bind to cynomolgus ROR2, to mouse ROR2 and to human ROR2 and comprise the variable domains of GK-5A1, MK-24C10 or MK-24F9, and preferably of GK-5A1.

In a preferred embodiment, the anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs of the invention originate from a mouse. The anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs of the invention may present favorable lower aggregation propensity, improved manufacturability and/or higher expression levels relative fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs obtained by methods involving phage display.

Also provided are polynucleotides encoding the fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof described herein, host cells transformed or transfected with these polynucleotides, and methods of making the various fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof described herein. The invention also provides substantially purified polynucleotides (DNA or RNA) that are identical or complementary to sequences encoding polypeptides comprising segments or domains of the antibody chains or antigen-binding fragments described herein. When expressed from appropriate expression vectors, polypeptides encoded by these polynucleotides are capable of exhibiting antigen-binding capacity. Also provided in the invention are polynucleotides that encode at least one CDR region and usually all three CDR regions from the heavy or light chain of the fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof described herein. Some other polynucleotides encode all or substantially all of the variable region sequence of the heavy chain and/or the light chain of the exemplified antibodies. Because of the degeneracy of the code, a variety of nucleic acid sequences will encode each of the immunoglobulin amino acid sequences. The polynucleotides of the invention can encode only the variable region sequence of an exemplified antibody. They can also encode both a variable region and a constant region of the antibody. Some of polynucleotide sequences of the invention nucleic acids encode a mature heavy chain variable region sequence that is substantially identical (e.g., at least 80%, 90%, or 99%) to the mature heavy chain variable region sequence shown. Some other polynucleotide sequences encode a mature light chain variable region sequence that is substantially identical to the mature light chain variable region sequence shown. Some of the polynucleotide sequences encode a polypeptide that comprises variable regions of both the heavy chain and the light chain of one of the exemplified antibody. Some other polynucleotides encode two polypeptide segments that respectively are substantially identical to the variable regions of the heavy chain and the light chain of one of the exemplified antibodies.

The polynucleotide sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence encoding an exemplified functional antibody. Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859, 1981; and the solid support method of U.S. Pat. No. 4,458,066. Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DNA Amplification, H. A. Erlich (Ed.), Freeman Press, N.Y., NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif., 1990; Manila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

Also provided in the invention are expression vectors and host cells for producing the fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof described herein. Various expression vectors can be employed to express the polynucleotides encoding the functional antibody chains or binding fragments. Both viral-based and nonviral expression vectors can be used to produce the antibodies in a mammalian host cell. Nonviral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., Nat. Genet. 15:345, 1997). For example, nonviral vectors useful for expression of the antibody polynucleotides and polypeptides in mammalian (e.g., human) cells include pCEP4, pREP4, pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C (Invitrogen, San Diego, Calif.), MPSV vectors, and numerous other vectors known in the art for expressing other proteins. Other useful nonviral vectors include vectors either comprising Sleeping Beauty, PiggyBac and other transposon systems or sequences allowing the transposition of the vector components by said Sleeping Beauty, PiggyBac and other transposon systems. Useful viral vectors include vectors based on lentiviruses or other retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992

The choice of expression vector depends on the intended host cells in which the vector is to be expressed. Typically, the expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) that are operably linked to the polynucleotides encoding a functional antibody chain or antigen-binding fragment. In some embodiments, an inducible promoter is employed to prevent expression of inserted sequences except under inducing conditions. Inducible promoters include, e.g., tetracycline-inducable, arabinose, lacZ, metallothionein promoters or a heat shock promoter. Cultures of transformed or transfected host cells can be expanded under non-inducing conditions without biasing the population for coding sequences whose expression products are better tolerated by the host cells. In addition to promoters, other regulatory elements may also be required or desired for efficient expression of a functional antibody chain or fragment. These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences. In addition, the efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer may be used to increase expression in mammalian host cells.

The expression vectors may also provide a secretion signal sequence position to form a fusion protein with polypeptides encoded by inserted functional antibody sequences. More often, the inserted functional antibody sequences are linked to a signal sequences before inclusion in the vector. Vectors to be used to receive sequences encoding the functional antibody light and heavy chain variable domains sometimes also encode constant regions or parts thereof. Such vectors allow expression of the variable regions as fusion proteins with the constant regions thereby leading to production of intact antibodies or fragments thereof. Typically, such constant regions are human.

The host cells for harboring and expressing the functional antibody chains can be either prokaryotic or eukaryotic. In some preferred embodiments, mammalian host cells are used to express and produce the antibody polypeptides of the present invention. For example, they can be either a hybridoma cell line expressing endogenous immunoglobulin genes or a mammalian cell line harboring an exogenous expression vector. These include any normal mortal or normal or abnormal immortal animal or human cell. In addition to the cell lines exemplified herein, a number of other suitable host cell lines capable of secreting intact immunoglobulins are also known in the art. These include, e.g., the CHO cell lines, various HEK 293 cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed cells of the B-lineage and hybridomas. The use of mammalian tissue cell culture to express polypeptides is discussed generally in, e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y., 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and/or modulatable or regulatable. Useful promoters include, but are not limited to, EFla and human UbC promoters exemplified herein, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

Methods for introducing expression vectors containing the polynucleotide sequences of interest vary depending on the type of cellular host. For example, calcium chloride transformation or electroporation is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts (see generally Sambrook et al., supra). Other methods include, e.g., electroporation, calcium phosphate treatment, liposome-mediated transfection, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion to the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivo transduction. For long-term, high-yield production of recombinant proteins, stable expression will often be desired. For example, cell lines which stably express the antibody chains or binding fragments can be prepared using expression vectors of the invention which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth of cells that successfully express the introduced sequences in selective media. Resistant, stably transfected cells can be proliferated using tissue culture techniques appropriate for the cell type.

Invention Aspects Relating to Novel ADCs and AECs

The present invention also refers to antibody effector conjugates (AECs) or antibody drug conjugates (ADCs).

The term “antibody effector conjugates” (AEC) is used herein to describe liposome or labeling agents linked to a fully human anti-ROR2 antibody, antibody-based binding protein or antigen-binding fragment thereof as described herein. The labeling agent allows direct or indirect detection of the anti-ROR2 antibody or antigen-binding fragment to which it is attached. The liposome, as described in Bendas, BioDrugs, 15: 215-224, 2001, can allow for controlled delivery of an agent to diseased cells. In preparing a liposome conjugate, e.g., an immunoliposome, an agent such as a chemotherapeutic or other drug can be entrapped in the liposome for delivery to a target cell.

The term “antibody drug conjugates” (ADC) is used herein to describe cytotoxic and/or cytostatic agents (“toxin”) linked to a fully human anti-ROR2 antibody, antibody-based binding protein or antigen-binding fragment as described herein. Cytotoxic and/or cytostatic agents are any agent known to inhibit the growth of and/or inhibit the replication of and/or kill cells, particularly malignant cells. Covalent conjugates of small molecular weight cytotoxic and/or cytostatic agents (with a molecular weight preferably <2'500 Daltons) to antibodies or antigen-binding fragments specific for tumor cells, are powerful tools to specifically target cancer cells for their destruction.

In ADCs, the toxin payload can be conjugated non-site-specifically to the antibody, antibody-based binding protein or antibody fragment via lysine or cysteine amino acid side chains employing classical chemical linkers with maleimide functionality, or other chemical known in the art that can mediate conjugation to lysine or cysteine amino acid side chains. In the ADCs of the invention, the small molecular weight payload can also be conjugated site-specifically either by chemical, chemo-enzymatic, or enzymatic conjugations known in the art, like e.g. with bifunctional linkers, linkers allowing Pictet-Spengler chemistry on formyl-glycine forming enzyme modified antibodies, by glycan-remodeled antibodies, or by bacterial transglutaminase or sortase enzymes.

According to one embodiment. the antibody drug conjugate (ADC) has the general formula A-(L)n-(T)m, in which

-   -   A is the antibody, derivative, modified format or fragment as         described herein,     -   L is a linker,     -   T is a cytotoxic or cytostatic payload

and in which n and m are integers between >1 and <10

According to one embodiment. the antibody effector conjugate (AEC) has the general having the general formula A-(L)n-(T)m, in which

-   -   A is the antibody, derivative, modified format or fragment         according to any one of claims 1     -   L is a linker,     -   T is a label

and in which n and m are integers between >1 and <10

In a preferred embodiment, the linker comprises, or consists of, at least one selected from the group consisting of: an oligopeptide linker (including cleavable and non-cleavable oligopeptide linkers), a hydrazine linker, a thiourea linker, a self-immolative linker, a succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC) linker, a disulfide linker, a thioether linker, and/or a maleimide linker.

The maleimide linker optionally comprises cleavable spacers, that may be cleaved by changes in pH, redox potential and or specific intracellular enzymes.

The skilled person understands that further linkers may be suitable. Such linkers may be non-cleavable or may be cleaved by changes in pH, redox potential or specific intracellular enzymes. Cleavable oligopeptide linkers include protease-cleavable linkers. It is understood that the linker may comprise combinations of the above. For example, the linker may be a valine-citruline PAB linker.

In one embodiment, the linker has at least one of the following amino acid sequences: -LPXTGn-, -LPXAGn-, -LPXSGn-, -LAXTGn-, -LPXTGn-, -LPXTAn- or -NPQTGn-, with Gn being an oligo- or polyglycine with n being an integer between ≥1 and ≤21, An being an oligo- or polyalanine with n being an integer between ≥1 and ≤21, and X being any conceivable amino acid sequence.

In a preferred embodiment, the linker is conjugated to the C-terminus of at least one subdomain of the antibody, antibody, derivative, modified format or fragment.

In one embodiment, prior to conjugation

-   -   the antibody, derivative, modified format or fragment bears a         sortase recognition motif fused or conjugated to the C-terminus         of at least one subdomain thereof, and     -   the toxin or label comprises a glycine stretch with a length of         between ≥1 and ≤21 glycine residues, preferably with a length of         ≥2 and ≤5 glycine residues.

The cytotoxic agent of the ADC can be a plant, fungal, or bacterial molecule. In some embodiments, the cytotoxic agent for conjugation to the antibody or antigen-binding fragment of the invention is a small molecule cellular toxin, a peptide toxin, or a protein toxin. Many specific examples of these toxins are well known in the art. See, e.g., Dyba et al., Curr. Pharm. Des. 10:2311-34, 2004; Kuyucak et al., Future Med. Chem. 6:1645-58, 2014; Beraud et al., Inflamm. Allergy Drug Targets. 10:322-42, 2011; and Middlebrook et al., Microbiol. Rev. 48:199-221, 1984. In some embodiments, a therapeutic agent is conjugated to the antibody. For example, the therapeutic agent can be a maytansinoid (e.g., maytansinol or DM1 maytansinoid), a taxane, a calicheamicin, a cemadotin, a monomethylauristatin (e.g., monomethylauristatin E or monomethylauristatin F), a pyrrolobenzodiazepine (PBD), an indilino-benzodiazepine pseudodimer or an anthracycline. Therapeutic agents also include vincristine and prednisone. In various embodiments, the therapeutic agent that may be employed in the invention can be an antimetabolite (e.g., an antifolate such as methotrexate, a fluoropyrimidine such as 5-fluorouracil, cytosine arabinoside, or an analogue of purine or adenosine); an intercalating agent (for example, an anthracycline such as doxorubicin, PNU-159682, daunomycin, epirabicin, idarubicin, mitomycin-C, dactinomycin, or mithramycin, or other intercalating agents such as pyrrolobenzodiazepine); a DNA-reactive agent such as calicheamicins, tiancimycins, and other enediynes; a platinum derivative (e.g., cisplatin or carboplatin); an alkylating agent (e.g., nitrogen mustard, melphalan, chlorambucil, busulphan, cyclophosphamide, ifosfamide nitrosoureas or thiotepa); an RNA polymerase inhibitor such as α-amanitin; an antimitotic agent (e.g., a vinca alkaloid such as vincristine, or a taxoid such as paclitaxel or docetaxel); a topoisomerase inhibitor (for example, etoposide, teniposide, amsacrine, topotecan); a cell cycle inhibitor (for example, a flavopyridol); or a microbtubule agent (e.g., an epothilone, a tubulysine, a pre-tubulysine, discodermolide analog, or eleutherobin analog). A therapeutic agent can be a proteosome inhibitor or a topoisomerase inhibitor such as bortezomib, amsacrine, etoposide, etoposide phosphate, teniposide, or doxorubicin. Therapeutic radioisotopes include iodine (¹³¹I) yttrium (⁹⁰Y), lutetium (¹⁷⁷Lu), actinium (²²⁵Ac), praseodymium, astatine (At), rhenium (Re), bismuth (Bi or Bi), and rhodium (Rh). Antiangiogenic agents include linomide, bevacuzimab, angiostatin, and razoxane.

In a preferred embodiment of the ADC, the cytotoxic or cytostatic payload is at least one selected from the group consisting of, or a derivative of:

-   -   maytansinoids,     -   auristatins,     -   anthracyclins,     -   calcheamicins,     -   tubulysins     -   duocarmycins     -   radioisotopes     -   liposomes comprising a toxid payload     -   protein toxins     -   taxanes     -   indilino-benzodiazepine pseudodimers, and/or     -   pyrrolobenzodiazepines.

In a preferred embodiment of the ADC, the toxin is selected from PNU-159682 as described in Quintieri et al. (2005) and derivatives thereof, maytansine, monomethyl auristatin MMAE, and monomethyl auristatin MMAF. In a preferred embodiment of the ADC, the toxin, joined to the linker at its wavy line, is of formula (i), as described in WO 2016/102679:

In the embodiment where the toxin is of formula (i), it is preferred that the linker comprise an alkyldiamino group of the form NH₂—(CH₂)_(m)—NH₂, where m≥1 and ≤11, preferably m=2, such that one amino group is directly linked at the wavy line of formula (i) to form an amide bond. It is moreover preferred that the second amino group is linked to an oligopeptide linker, which is more preferably an oligoglycine.

In some embodiments, the molecule for conjugation to the antibody is a protein (e.g., an antibody) or an RNA or DNA aptamer.

In a preferred embodiment of the AEC, the label is at least one selected from the group consisting of: a fluorescent label (including a fluorescent dye or a fluorescent protein), a chromophore label, a radioisotope label containing iodine (e.g., ¹²⁵I), gallium (⁶⁷Ga), indium (¹¹¹I), technetium (^(99m)Tc), phosphorus (³²P), carbon (¹⁴C), tritium (³H), other radioisotope (e.g., a radioactive ion), and/or a protein label such as avidin or streptavidin.

In a preferred embodiment of the ADC or AEC, the ADC or AEC has a stoichiometric ratio between antibody and payload of any integer between ≥1 and ≤10, preferably 2 and 4. In the case of the ADC, this ratio may also be referred to as the drug to antibody ratio (“DAR”). In the case of a mixture or collection of ADCs, the DAR may be a rational number representing the average of the individual integer DARs.

In one embodiment of the ADC or AEC, only one or more heavy chains of the antibody or antigen-binding fragment according are conjugated to a payload.

In one embodiment of the ADC or AEC, only one or more light chains of the antibody or antigen-binding fragment according are conjugated to a payload.

It is understood that a given ADC or AEC may comprise different toxins or labels bound simultaneously; it is also possible that the anti-ROR2 antibody be linked to a combination of both one or more labels and one or more toxins.

The antibodies or antigen-binding fragments of the invention can be conjugated to a synthetic molecule using any type of suitable conjugation. Recombinant engineering and incorporated selenocysteine (e.g., as described in U.S. Pat. No. 8,916,159) can be used to conjugate a synthetic molecule. Other methods of conjugation can include covalent coupling to native or engineered lysine side-chain amines or cysteine side-chain thiols. See, e.g., Wu et al., Nat. Biotechnol, 23: 1 137-1 146 (2005). The synthetic molecule can be any molecule such as one targeting a tumor.

In a preferred embodiment, the ADC or AEC is obtained by means of site-specific sortase-enzyme mediated antibody conjugation (SMAC-technology), which is enablingly disclosed in WO2014140317 assigned to the same applicant. The content of this publication is incorporated by reference herein. Sortases (also called sortase transpeptidases) form a group of prokaryotic enzymes that modify surface proteins by recognizing and cleaving a specific peptide motif called “sortase recognition motif” or “sortase tag”. Usually, a given sortase enzyme recognizes one or more sortase recognition motifs. Sortase enzymes can be naturally occurring, or may have undergone genetic engineering (Dorr et al., 2014), and are used, in the SMAC technology, to conjugate two proteins one of which is bearing such recognition motif, while the other one is bearing a oligo-glycine peptide (Gly)_(n). It is important to understand that, in one specific embodiment (where Streptococcus pyogenes sortase A is used, see below), the oligo-glycine (Gly)_(n) can optionally be replaced by an oligo-alanine (Ala)_(n).

In a preferred embodiment, the ADC or AEC is obtained by means of site-specific sortase-enzyme mediated conjugation of:

-   a) an antibody or antigen-binding fragment as described herein     carrying one or more sortase recognition motifs, and -   b) one or more payloads carrying an oligoglycine tag; -   or -   a) an antibody or antigen-binding fragment as described herein     carrying one or more oligoglycine tags, and -   b) one or more payloads carrying a sortase recognition motif.

It is important to understand that, in one specific embodiment (where Streptococcus pyogenes sortase A is used, see below), the oligo-glycine (Gly)_(n) can optionally be replaced by an oligo-alanine (Ala)_(n).

Preferably, the sortase recognition motif is fused or conjugated to the C-terminus of at least one subdomain of the antibody.

Preferably, the oligo glycine tag has a length between ≥1 and ≤21 glycine residues, preferably with a length between ≥2 and ≤5 amino acids.

The invention also refers to a method of producing an AEC or ADC, which method comprises the following steps:

-   -   a) providing an antibody, derivative, modified format or         fragment as described herein, which antibody or antigen-binding         fragment carries one or more sortase recognition motifs,     -   b) providing one or more payloads carrying an oligoglycine tag,         and     -   c) conjugating the antibody or antigen-binding fragment and one         or more payloads by means of sortase-mediated conjugation.

The term “oligoglycine tag” (also called Gn or G(n)) relates to an oligoglycine the length n of which can be between ≥1 and ≤21, preferably between ≥1 and ≤5.

The invention also refers to a method of producing an AEC or ADC, which method comprises the following steps:

-   -   a) providing an anti-ROR2 antibody or antigen-binding fragment         as described herein, which antibody or antigen-binding fragment         carries one or more oligoglycine tags,     -   b) providing one or more payloads carrying a sortase recognition         motif, and     -   c) conjugating the antibody or antigen-binding fragment and one         or more payloads by means of sortase-mediated conjugation.

According to another embodiment, said sortase enzyme recognition motif comprises at least one of the following amino acid sequences: LPXTG, LPXAG, LPXSG, LAXTG, LPXTA or NPQTN, with X being any conceivable amino acid sequence.

According to another embodiment, the resulting linker has at least one of the following amino acid sequences: -LPXTGn-, -LPXAGn-, -LPXSGn-, -LAXTGn-, -LPXTGn-, -LPXTAn- or -NPQTGn-, with Gn being an oligo- or polyglycine with n being an integer between ≥1 and ≤21, An being an oligo- or polyalanine with n being an integer between ≥1 and ≤21, and X being any conceivable amino acid sequence.

The following table shows the recognition tags and the peptides derived therefrom to be part of the linker:

TABLE 4 Sortase recognition motifs and resulting linkers Staphylococcus aureus sortase A recognition -LPXTG sequence, with X being any amino acid Staphylococcus aureus sortase A recognition -LPXAG sequence, with X being any amino acid recognition sequence for Staphylococcus aureus -LPXSG sortase A or engineered sortase A 4S-9 from Staphylococcus aureus, with X being any amino acid recognition sequence for engineered sortase A -LAXTG 2A-9 from Staphylococcus aureus, with X being any amino acid Streptococcus pyogenes sortase A recognition -LPXTA sequence, with X being any amino acid Staphylococcus aureus sortase recognition -NPQTN sequence Linker derived from Staphylococcus aureus -LPXT(Gn)- sortase A recognition sequence, with X being any amino acid and n ≥ 1 and ≤21 Linker derived from Staphylococcus aureus -LPXA(Gn)- sortase A recognition sequence, with X being any amino acid and n ≥ 1 and ≤21 Linker derived from recognition sequence for -LPXS(Gn)- Staphylococcus aureus sortase A or engineered sortase A 4S-9 from Staphylococcus aureus, with X being any amino acid and n ≥ 1 and ≤21 Linker derived from recognition sequence for -LAXT(Gn)- engineered sortase A 2A-9 from Staphylococcus aureus, with X being any amino acid and n ≥ 1 and ≤21 Linker derived from Streptococcus pyogenes -LPXT(Gn)- or sortase A recognition sequence, with X being -LPXT(An)- any amino acid and n ≥ 1 and ≤21 Linker derived from Staphylococcus aureus -NPQT(Gn)- sortase recognition sequence, with n ≥ 1 and ≤21

Engineered sortases, including but not limited to sortase A mutant 2A-9 and sortase A mutant 4S-9 from Staphylococcus aureus, are described in Don et al. (2014) and mutants described in Chen et al. (2011).

As background and to exemplify the general concept of sortase transpeptidation, Sortase A uses an oligo-glycine-stretch as a nucleophile to catalyze a transpeptidation by which the terminal amino group of the oligo-glycine effects a nucleophilic attack on the peptide bond joining the last two C-terminal residues of the sortase tag. This results in breakage of that peptide bond and formation of a new peptide bond between the C-terminally second-to-last residue of the sortase tag and the N-terminal glycine of the oligo-glycine peptide, i.e. resulting in a transpeptidation.

It is important to understand that, in one specific embodiment (where Streptococcus pyogenes sortase A is used, see above), the oligo-glycine (Gly)n can optionally be replaced by an oligo-alanine (Ala)n.

Prior to sortase conjugation, the sortase recognition motif may, at its C-terminus, furthermore carry other tags, like His-tags, Myc-tags or Strep-tags (see FIG. 4a of WO 2014/140317, the content of which is incorporated by reference herein). However, because the peptide bond between the 4th and 5th amino acid of the sortase tag is cleaved upon sortase A mediated conjugation, these additional tags do not appear in the conjugated product.

The sortase tag may, for example, be fused to a C-terminus of a binding protein, or to a domain or subunit thereof, by genetic fusion and co-expressed therewith. In another preferred embodiment, the sortase tag may directly be appended to the last naturally occurring C-terminal amino acid of the immunoglobulin light chains or heavy chains, which in case of the human immunoglobulin kappa light chain is the C-terminal cysteine residue, and which in the case of the human immunoglobulin IgG1 heavy chain may be the C-terminal lysine residue encoded by human Fcγ1 cDNA. However, another preferred embodiment is also to directly append the sortase tag to the second last C-terminal glycine residue encoded by human Fcγ1 cDNA, because usually terminal lysine residues of antibody heavy chains are clipped off by posttranslational modification in mammalian cells. Therefore, in more than 90% of the cases naturally occurring human IgG1 lacks the C-terminal lysine residues of the IgG1 heavy chains. Therefore, one preferred embodiment of the invention is to omit the C-terminal lysine amino acid residues of human IgG1 heavy chain constant regions in expression constructs for sortase recognition-motif tagged Igyl heavy chains. Another preferred embodiment is to include the C-terminal lysine amino acid residues of human IgG1 heavy chain constant regions in expression constructs for sortase recognition-motif tagged Igyl heavy chains.

In another preferred embodiment the sortase or oligoglycine tag may be appended to the C-terminus of a human immunoglobulin IgG1 heavy chain where the C-terminal lysine residue encoded by human Fcγ1 cDNA is replaced by an amino acid residue other than lysine to prevent unproductive reactions of sortase with the ε-amino group of said C-terminal lysine residue leading to inter-heavy chain crosslinking.

We have described previously that in some cases (e.g. at the C-terminus of the Ig kappa light chains, see: Beerli et al. (2015) PloS One 10, e131177) it is beneficial to add additional amino acids between the C-terminus of the binding protein and the sortase tag. This has been shown to improve sortase enzyme conjugation efficiencies of payloads to the binding protein. In the case of Ig kappa light chains, it was observed that by adding 5 amino acids between the last C-terminal cysteine amino acid of the Ig kappa light chain and the sortase pentapeptide motif improved the kinetic of conjugation, so that the C-termini of Ig kappa light chains and Ig heavy chains could be conjugated with similar kinetics (see: Beerli et al. (2015) PloS One 10, e131177). Therefore, it is another preferred embodiment that optionally ≥1 and ≤11 amino acids are added in between the last C-terminal amino acid of a binding protein or antibody subunit and the sortase tag.

In a preferred embodiment, a GnS peptide (wherein n is between ≥1 and ≤21, preferably between ≥1 and ≤5 is added between the last C-terminal amino acid of a binding protein or antibody subunit and the sortase tag.

Finally, in another preferred embodiment, additional amino acids between the C-terminus of the binding protein and the sortase or oligoglycine tag may beneficially be included that comprise a sequence and/or linker that is cleavable by hydrolysis, by a pH change or by a change in redox potential, or that is cleavable by a non-sortase enzyme, e.g., by proteases.

Acording to another aspect of the invention, a ROR2 chimeric antigen receptor (CAR) employing the antibody, derivative, modified format or fragment according to the above description is provided, fused or conjugated to at least one transmembrane region and at least one intracellular domain.

Further, a cell comprising such a chimeric antigen receptor is provided, which cell is preferably an engineered T-cell.

Further, the use of the antibody, derivative, modified format or fragment, the antibody drug conjugate or the CAR or cell according to the above description for the treatment of a patient that is

-   -   suffering from,     -   at risk of developing, and/or     -   being diagnosed for

a neoplastic disease is provided.

As an alternative, a method of treating a patient suffering from, at risk of developing, and/or being diagnosed for a neoplastic disease is provided, which method comprises the administration of one or more therapeutically active doses of the antibody, the antibody drug conjugate or the CAR or cell according to the above description.

In one embodiment, the neoplastic disease is at least one selected from the group consisting of

-   -   renal cell carcinoma     -   osteosarcoma     -   kidney cancer

According to one aspect, a pharmaceutical composition comprising the antibody, derivative, modified format or fragment, the antibody drug conjugate or the CAR or cell according to the above description is provided, together with one or more pharmaceutically acceptable ingredients.

In another aspect, the invention provides methods of killing or inhibiting the growth of a cell expressing ROR2 in vitro or in a patient is provided, which method comprises administering to the cell a pharmaceutically effective amount or dosis of the antibody, derivative, modified format or fragment, the antibody drug conjugate or the CAR or cell according to the above description or the pharmaceutical composition according to the above description.

Preferably, the cell expressing ROR2 is a cancer cell.

The methods entail administering a therapeutically effective amount of an anti-ROR2 antibody, antibody-based binding protein, antibody fragment thereof, ADC or CAR disclosed herein to a subject in need of treatment. This enables killing or inhibiting the growth of the cell expressing ROR2 in the subject. In various embodiments, the cell expressing ROR2 is a tumor cell. In a related aspect, the invention provides methods of treating a disease or condition associated with elevated expression of ROR2 in a subject. These methods involve administering a therapeutically effective amount of an anti-ROR2 antibody, antibody-based binding protein, antibody fragment thereof, ADC or CAR of the invention to a subject that is afflicted with a disease or condition associated with elevated expression of ROR2. This enables treatment of the disease or condition in the subject. Some of these methods are directed to treating a cancer in the subject. Cancers that are amendable to treatment with methods of the invention include, e.g., neuroblastoma, sarcoma (and especially osteosarcoma), renal cell carcinoma, breast cancer, testicular cancer, ovarian cancer, pancreatic cancer, kidney cancer, renal cancer, gastric cancer, prostate cancer, head and neck cancer, melanoma, squamous cell carcinoma, multiple myeloma and other cancers.

In various embodiments, the administered anti-ROR2 antibody, antibody-based binding protein, antibody fragment thereof, or ADC or CAR based thereon, are F(ab)2, Fv, scFv, IgGACH2, F(ab′)2, scFv2CH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, (scFv)2, or a synthetic IgG. In some embodiments, the administered anti-ROR2 antibody, antibody-based binding protein or antibody fragment thereof is conjugated to a synthetic molecule. In some of these embodiments, the anti-ROR2 antibody, antibody-based binding protein, antibody fragment thereof is conjugated to a transmembrane region and an intracellular T-cell receptor (TCR) signaling domain to form a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor (CAR) is present on a T cell to be administered to the subject. In some other embodiments, the antibody can be conjugated to a cytotoxic agent, a radioisotope, or a liposome.

The terms “treating” or “treatment” used herein do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment recognized by one of ordinary skill in the art as having a potential benefit or therapeutic effect. In this respect, the inventive method can provide any amount of any level of treatment. Furthermore, the treatment provided by the inventive method can include the treatment of one or more conditions or symptoms of the disease being treated. In particular, the treatment may be administered as an intravenous infusion.

In one embodiment, the fully human anti-ROR2 antibody, antibody-based binding protein or antibody fragment thereof, bi- or multispecific antibody, ADC or CAR is administered as a monotherapy. In an alternative embodiment, the fully human anti-ROR2 antibody, antibody-based binding protein or antibody fragment thereof, bi- or multispecific antibody, ADC or CAR is administered as with or in parallel or series to further therapeutic agents.

In particular, the fully human anti-ROR2 antibody antibody-based binding protein or antibody fragment thereof, bi- or multispecific antibody, ADC or CAR may be administered at a dosage of about 0.1-20 mg/kg.

The term “subject” refers to human and non-human animals (especially non-human mammals), and preferably to human animals. In spite of the antibody, antibody-based binding protein or antibody fragment thereof, bi- or multispecific antibody, ADC or CAR binding to ROR2, they may also bind to ROR2 from other species making them effective for treatment of these species.

The present invention thus also refers to a method for treating a subject suffering from, being at risk of developing, and/or being diagnosed with a neoplastic disease, comprising administration of a fully human anti-ROR2 antibody, antibody-based binding protein or antibody fragment thereof, ADC or CAR, as described herein.

In particular, said neoplastic disease is selected from the group consisting of: neuroblastoma, sarcoma (and especially osteosarcoma), renal cell carcinoma, breast cancer, testicular cancer, ovarian cancer, pancreatic cancer, kidney cancer, renal cancer, gastric cancer, prostate cancer, head and neck cancer, melanoma, squamous cell carcinoma, multiple myeloma and other cancers.

In some related aspects, the invention provides pharmaceutical compositions or kits that contain a therapeutically effective amount of a fully human anti-ROR2 antibody or antibody, antibody-based binding protein or antibody fragment thereof, bi- or multispecific antibody, or ADC described herein and a pharmaceutically acceptable carrier. Some kits of the invention can additionally contain one or more immunoassay buffers.

In some embodiments, the compositions of the invention contain a carrier, desirably a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be one or more compatible solid or liquid fillers, diluents, other excipients, or encapsulating substances which are suitable for administration into a human or veterinary subject (e.g., a physiologically acceptable carrier or a pharmacologically acceptable carrier). The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the use of the active ingredient, e.g., the administration of the active ingredient to a subject. The pharmaceutically acceptable carrier can be co-mingled with one or more of the active components, e.g., a hybrid molecule, and with each other, when more than one pharmaceutically acceptable carrier is present in the composition, in a manner so as not to substantially impair the desired pharmaceutical efficacy. Pharmaceutically acceptable materials typically are capable of administration to a subject, e.g., a patient, without the production of significant undesirable physiological effects such as nausea, dizziness, rash, or gastric upset. It is, for example, desirable for a composition comprising a pharmaceutically acceptable carrier not to be immunogenic when administered to a human patient for therapeutic purposes.

Pharmaceutical compositions of the invention can additionally contain suitable buffering agents, including, for example, acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt. The compositions can also optionally contain suitable preservatives, such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal. Pharmaceutical compositions of the invention can be presented in unit dosage form and can be prepared by any suitable method, many of which are well known in the art of pharmacy. Such methods include the step of bringing the antibody or antigen-binding fragment of the invention into association with a carrier that constitutes one or more accessory ingredients. In general, the composition is prepared by uniformly and intimately bringing the active agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

A composition suitable for parenteral administration conveniently comprises a sterile aqueous preparation of the inventive composition, which preferably is isotonic with the blood of the recipient. This aqueous preparation can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also can be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed, such as synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.

Preparation of pharmaceutical compositions of the invention and their various routes of administration can be carried out in accordance with methods well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. The delivery systems useful in the context of the invention include time-released, delayed release, and sustained release delivery systems such that the delivery of the inventive composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. The inventive composition can be used in conjunction with other therapeutic agents or therapies. Such systems can avoid repeated administrations of the inventive composition, thereby increasing convenience to the subject and the physician, and may be particularly suitable for certain compositions of the invention.

Many types of release delivery systems are available and known to those of ordinary skill in the art. Suitable release delivery systems include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer systems that are lipids including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di- and triglycerides; hydrogel release systems; sylastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the active composition is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034, and 5,239,660 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,832,253 and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

The invention also provides kits suitable for carrying out the methods of the invention. Typically, the kits contain two or more components required for performing the therapeutic or diagnostic methods of the invention. Kit components include, but are not limited to, one or more antibodies or antigen-binding fragments of the invention, appropriate reagents, and/or equipment. In some embodiments, the kits can contain an antibody or antigen-binding fragment of the invention and an immunoassay buffer suitable for detecting ROR2 (e.g. by ELISA, flow cytometry, magnetic sorting, or FACS). The kit may also contain one or more microtiter plates, standards, assay diluents, wash buffers, adhesive plate covers, magnetic beads, magnets, and/or instructions for carrying out a method of the invention using the kit. The kit can include an antibody or antigen-binding fragment of the invention bound to a substrate (e.g., a multi-well plate or a chip), which is suitably packaged and useful to detect ROR2. In some embodiments, the kits include an antibody or antigen-binding fragment of the invention that is conjugated to a label, such as, a fluorescent label, a biologically active enzyme label, a luminescent label, or a chromophore label. The kits can further include reagents for visualizing the conjugated antibody, e.g., a substrate for the enzyme. In some embodiments, the kits include an antibody or antigen-binding fragment of the invention that is conjugated to a contrast agent and, optionally, one or more reagents or pieces of equipment useful for imaging the antibody in a subject.

Generally the antibody or antigen-binding fragment of the invention in a kit is suitably packaged, e.g., in a vial, pouch, ampoule, and/or any container appropriate for a therapeutic or detection method. Kit components can be provided as concentrates (including lyophilized compositions), which may be further diluted prior to use, or they can be provided at the concentration of use. For use of the antibody antibody, derivative, modified format or fragment of the invention in vivo, single dosages may be provided in sterilized containers having the desired amount and concentration of components.

Invention aspects relating to methods of detection

According to another aspect of the invention, a method of determining whether a suspected patient is

-   -   suffering from,     -   at risk of developing, and/or     -   diagnosed for

a neoplastic disease or immune disease is provided, said method comprising the treatment of a sample taken from that subject with an antibody effector conjugate according to the abive description.

In one embodiment, the neoplastic disease or immune disease is suitable for treatment with an anti ROR2 antibody, anti ROR2 antibody drug conjugate, or a CAR T cell comprising an anti ROR2 antibody.

In some other embodiments, the invention provides method for detecting in a biological sample an altered level of ROR2 (e.g., cell surface ROR2), for example, relative to a control, either by FACS, immunohistochemistry (IHC) or Western Blotting. Generally, the method includes contacting a biological sample with an antibody, antibody-based binding protein, antibody fragment thereof of the invention and determining the amount of antibody that selectively binds to material (e.g., cells) in the sample to thereby determine the level of ROR2 in the biological sample. A biological sample can be from a cell culture or from a test subject, e.g., a plasma or a tissue sample from a subject that has, is suspected to have, or is at risk of developing a disease or condition associated with elevated ROR2 in a subject. A control level desirably corresponds to the ROR2 level detected using the same antibody in a corresponding sample(s) from one or more control cultures or disease-free subjects. Methods of using the antibody of the invention to determine ROR2 levels can include any immunoassay such as immuno- (Western) blotting, enzyme-linked immunosorbent assay (ELISA), Immunohistochemistry (IHC) and flow cytometry, e.g., fluorescence-activated cell sorting (FACS) analysis.

The methods of detection can be used to screen for the presence of a disorder associated with elevated ROR2. The methods include obtaining a sample from a test subject in need of screening, e.g., a subject that has, is suspected to have, or is at risk of developing a disorder associated with elevated ROR2. The level of ROR2 (e.g., the amount or concentration) in the sample is measured using an antibody, antibody-based binding protein, antibody fragment thereof of the invention, and the level in the sample is compared to a control level of ROR2. The control level represents, for example, the mean level (e.g., the amount or concentration) in sample(s) from one or, preferably, multiple control group subjects that do not have a disorder associated with elevated ROR2. Alternatively, the control level can correspond to the level or mean level of ROR2 in one or more samples taken from the test subject at one or more prior times, such as when the test subject did not have or did not exhibit, a condition associated with elevated ROR2. A significantly higher level of ROR2 in the biological sample relative to the control level is indicative of a disorder associated with elevated ROR2 in the subject. In subjects such as humans, where cell surface ROR2 expression is largely restricted to embryonic development, a control level of ROR2 can be zero or none. Thus, in some embodiments of the method of detection provided by the invention, any significant and detectable amount of ROR2 in a biological sample can be indicative of a disorder associated with elevated ROR2 in the subject.

Additionally, the methods of detection can be used to monitor the progress of a disorder associated with elevated ROR2. The method includes obtaining a sample from a subject in need of screening, e.g., a subject having been diagnosed or suspected to have a disorder associated with elevated ROR2. The level of ROR2 in the sample is measured using an antibody, antibody-based binding protein, antibody fragment thereof of the invention, and the level in the sample is compared to a control level corresponding to the level or mean level of ROR2 in one or more samples taken from the test subject at one or more prior times. Levels of ROR2 that are significantly elevated or decreased relative to control indicate that the subject's disorder is deteriorating or improving, respectively. The foregoing methods of detection can be used to screen for the presence or to monitor the progress of disorders including, for example, CLL, ALL, mantle cell lymphoma, neuroblastoma, sarcoma, renal cell carcinoma, breast cancer, lung cancer, colon cancer, head and neck cancer, melanoma, and other cancers.

In some embodiments, the invention provides methods for screening a subject for an altered level of ROR2. Generally, the methods entail administering to the subject an antibody, antibody-based binding protein, antibody fragment thereof of the invention that is conjugated to a label (e.g., a contrast agent), imaging the subject in a manner suitable for detecting the label, and determining whether a region in the subject has an altered density or concentration of label as compared to the background level of label in proximal tissue. Alternatively, the methods include determining whether there is an altered density or concentration of label in a region as compared to the density or concentration of label previously detected in the same region of the subject. Methods of imaging a subject can include x-ray imaging, x-ray computed tomography (CT) imaging (e.g., CT angiography (CTA) imaging), magnetic resonance (MR) imaging, magnetic resonance angiography (MRA), nuclear medicine, ultrasound (US) imaging, optical imaging, elastography, infrared imaging, microwave imaging, and the like, as appropriate for detecting the label conjugated to the antibody. In a preferred embodiment, the subject has, is suspected to have, or is at risk of developing an ROR2-expressing tumor, such as CLL, ALL, mantle cell lymphoma, neuroblastoma, sarcoma, renal cell carcinoma, breast cancer, lung cancer, colon cancer, head and neck cancer, melanoma, and other cancers, and the method is used to screen for or detect the presence of the tumor. In another embodiment, the method can be used to monitor the size or density of a ROR2-expressing tumor over time, e.g., during a course of treatment.

The invention thus provides methods for detecting an altered ROR2 level in a subject. The methods involve (a) obtaining a biological sample from the subject, (b) contacting the sample with an anti-ROR2 antibody, antibody-based binding protein, antibody fragment thereof disclosed herein, (c) determining the level of ROR2 in the biological sample, and (d) comparing the level of ROR2 in the biological sample to a control level of ROR2. This allows determination of whether the ROR2 level in the biological sample is altered relative to the control level of ROR2. In these methods, an increased ROR2 level in the subject relative to the control level is indicative of a disease or condition associated with elevated expression of ROR2 in the subject. For example, detection of elevated ROR2 expression can be indicative of the presence of neuroblastoma, osteosarcoma, renal cell carcinoma, breast cancer, gastric cancer, prostate cancer, melanoma, squamous cell carcinoma, or multiple myeloma in the subject.

In still another aspect, the invention provides methods for detecting a ROR2-expressing tumor in a subject. These methods entail (a) administering an anti-ROR2 antibody, antibody-based binding protein, antibody fragment thereof of the invention to a subject that has, is suspected to have, or is at risk of developing an ROR2-expressing tumor, and (b) imaging the subject for a region of altered conjugated label density or concentration, wherein the density or concentration is relative to (i) background in proximal tissue or (ii) the density or concentration previously detected in the same region of the subject. The existence of a region of altered conjugated label density or concentration is an indication of the presence of a ROR2-expressing tumor in the subject.

Invention aspects relating cells and T cells

In some embodiments, the invention provides methods for treating a subject that has, is suspected to have, or is at risk of developing a disorder associated with elevated levels of ROR2 by adoptive transfer of the genetically engineered T-cells described herein, which express an antibody or antigen-binding fragment of the invention as a chimeric antigen receptor (CAR) that selectively binds ROR2. Recombinant technology can be used to introduce CAR-encoding genetic material into any suitable T-cells, e.g., central memory T-cells from the subject to be treated. The T cells carrying the genetic material can be expanded (e.g., in the presence of cytokines). The genetically engineered T-cells are transferred, typically by infusion, to the patient. The transferred T-cells of the invention can then mount an immune response against ROR2 expressing cells in the subject. The adoptive transfer method can be used, for example, to treat subjects that have or are suspected to have any of the cancers associated with ROR2, including CLL, ALL, mantle cell lymphoma, neuroblastoma, sarcoma, renal cell carcinoma, breast cancer, lung cancer, colon cancer, head and neck cancer, melanoma, and other cancers. In some embodiments, the foregoing methods of treatment can further include co-administering a second therapeutic agent for treating the disorder associated with elevated ROR2. For example, when the disorder to be treated involves an ROR2-expressing cancer, the method can further include co-administration of a cytotoxic, cystostatic, or antiangiogenic or immune-stimulatory agent (e.g. immune-checkpoint inhibitor antibodies, for instance, but not limited to, those binding to PD1, PDL1, CTLA4, OX40, TIM3, GITR, LAG3 and the like) suitable for treating the cancer. If the cancer is a B-cell malignancy, the method can further include, for example, co-administration of rituximab, alemtuzumab, ofatumumab, ocrelizumab, or a CHOP chemotherapeutic regimen.

The invention thus further provides eukaryotic or non-eukaryotic cells (e.g., T lymphocytes) that have been recombinantly engineered to produce the fully human anti-ROR2 antibodies or antigen-binding fragments of the invention. The eukaryotic or non-eukaryotic cells can be used as an expression system to produce the antibody of the invention. In some embodiments, the invention provides ROR2 targeted immune cells that are engineered to recombinantly express a ROR2-specific non-human or humanized antibody or antigen-binding fragment of the invention. For example, the invention provides a T cell engineered to express a human anti-ROR2 antibody or antigen-binding fragment of the invention (e.g., an scFv, scFv-Fc, or (scFv)2), which is linked to a synthetic molecule containing one or more of the following domains: a spacer or hinge region (e.g., a CD28 sequence or a IgG4 hinge-Fc sequence), a transmembrane region (e.g., a transmembrane canonical domain), and an intracellular T-cell receptor (TCR) signaling domain, thereby forming a chimeric antigen receptor (CAR) or T-body. Intracellular TCR signaling domains that can be included in a CAR (or T-body) include, but are not limited to, CD3ζ, FcR-γ, and Syk-PT signaling domains as well as the CD28, 4-1BB, and CD134 co-signaling domains. Methods for constructing T-cells expressing a CAR (or T-body) are known in the art. See, e.g., Marcu-Malina et al., Expert Opinion on Biological Therapy, Vol. 9, No. 5 (posted online on Apr. 16, 2009).

EXAMPLES

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5′→3′.

Example 1: Antibody Library Generation and Screening (FIG. 1)

hROR2-TwinStrep Antigen Expression.

The EBNA expression vector pCB14b-hROR2-Thr-ECD-TwinStrep (where Thr is threonine), directing expression of the hROR2 extracellular domain (ECD), C-terminally tagged with a TwinStrep tag, was transfected into HEK293T using Lipofectamine® LTX with PLUS™ Reagent (Thermo Fisher Scientific, 15388100). Following a 1-day incubation (37° C., 5% CO₂, growth media: Dulbecco's Modified Eagle Medium (DMEM) High Glucose (4.5 g/L) with L-Glutamine with 10% (v/v) Fetal Calf Serum (FCS), 100 IU/mL of Pen-Strep-Fungizone and 2 mM L-glutamine (all Bioconcept)), cells were expanded under selection conditions (2 μg/mL of puromycin (Sigma-Aldrich, P8833-25 mg stock at 2 mg/mL)). Cells were split and further expanded (37° C., 5% CO₂); once confluency was reached, hyperflasks were coated with 20 μg/ml poly-L-Lysine (Sigma-Aldrich, P1524) for 2 hrs at 37° C. and washed twice with PBS. Then, cells were trypsinized, washed with PBS and plated onto poly-L-lysine-coated hyperflasks. After reaching confluency again, cells were washed with PBS followed by media replacement using production media (DMEM/F-12, Gibco/Thermo Fisher Scientific, 31330-038) supplemented with 1 μg/mL puromycin (Sigma-Aldrich, P8833), 100 IU/mL of Pen-Strep-Fungizone (Bioconcept, 4-02F00-H), 161 μg/mL of N-acetyl-L-cysteine (Sigma-Aldrich, A8199) and 10 μg/mL of L-glutathione reduced (Sigma-Aldrich, G6529). Supernatant, harvested bi-weekly and filtered (0.22 μm) to remove cells, was stored at 4° C. until purification. Removed supernatant was replaced with fresh production medium. ExpressPAGE of all harvests confirmed the presence of bands corresponding hROR2.

For purification, filtered supernatant was loaded onto a column suitable for binding strep tags; purification and elution was performed according to the manufacturer's protocol on an AEKTA pure (GE Healthcare). Fractions were analyzed for protein purity and integrity by SDS-PAGE. Protein-containing fractions were mixed and subjected to buffer exchange using Amicon filtration units (Millipore, Schaffhausen, Switzerland, UFC901008) to reach a dilution of ≥1:100 in PBS, and then sterile filtered using a low retention filter (0.20 μm, Carl Roth, Karlsruhe, Germany, Pa.49.1).

H2L2 Mouse Immunization with hROR2-TwinStrep.

Five transgenic humanized H2L2 mice (obtained from Harbour Biomed; H2L2 mice are a cross of the following mouse strains: F129, fvb/n and C57BL6, and on immunization produce antibodies with a human variable domain and a rat constant domain, disclosed in WO 2010/070263 A1), aged 6-10 weeks, were each immunized by two intraperitoneal (IP) injections followed by one intravenous injection of 100 μL of hROR2-TwinStrep (from Example 1), formulated as per Table 5.

TABLE 5 H2L2 mouse immunization schedule hROR2-Twin Injected Day Strep quantity Formulation volume 0 50 μg 20 μg monophosphoryl lipid 100 μL (MPLA, Sigma tlrl-mpls) and a 50:50 by volume mixture of Addavax adjuvant (Sigma, vac-adx-10) in PBS to reach 100 μL 21 20 μg 20 μg monophosphoryl lipid 100 μL (MPLA, Sigma tlrl-mpls) and a 50:50 by volume mixture of Addavax adjuvant (Sigma, vac-adx-10) in PBS to reach 100 μL 42 10 μg PBS 100 μL

Blood samples were collected from each mouse by tail bleed on days: −7, 7 and 28 relative to the first injection, and by heart puncture on day 49. All procedures herein described involving animals were compliant with Swiss guidelines on animal care and handling.

Determination of Antibody Titers.

Collected blood samples were incubated at room temperature for 15-60 minutes to allow clotting of the blood and subsequently centrifuged to obtain serum, in which antibody titers were evaluated. For this, ELISA plates were coated with 100 μL of 2 μg/ml hROR2-TwinStrep (from Example 1) in sodium bicarbonate coating buffer (0.1M Na₂CO₃, 0.1M NaHCO₃, pH 9.6). Following 1-day incubation at 4° C., plates were washed with PBS supplemented with 0.05% (v/v) of Tween 20 and blocked with 150 μL of PBS supplemented with 0.05% (v/v) of Tween 20 and 3% of bovine serum albumin (BSA). Serum samples were diluted 100-fold, and a 2.5-fold dilution series prepared therefrom, adding 50 μL of each dilution to respective ELISA plate wells. Following 1 h incubation at 37° C., plates were washed with PBS supplemented with 0.05% (v/v) of Tween 20 before addition of HRP-conjugated anti-rat FC gamma fragment (Jackson Immunoresearch, 112-036-071). Following further plate washing, plates were developed with 504, of Sigmafast OPD Tablet set (Sigma, P9187) and the reaction stopped by addition of 2M sulfuric acid. Plates were read on an ELISA plate reader (OD 490), with results given in FIG. 2.

MACS Isolation of hROR2 Antigen Specific B-Cells from H2L2 Mice.

Mice were euthanized at 49-days following initial antigen injection, and mouse spleens were harvested. Spleens were transferred into a gentleMACS C-tube (cat. Nr. 130-093-237) containing 2.4 ml RPMI-10% FCS, and homogenized using the gentleMACS Octo Dissociator (Miltenyi Biotec) prior to filtration through a cell strainer (FACS tubes, BD Flacon, 734-0001) to get single cell suspensions.

Anti-hROR2-specific B cells from mouse splenocytes were then selected by MACS sorting: first, B cells were isolated using a mouse Pan B Cell Isolation Kit, human (Miltenyi Biotec, 130-095-813) by negative selection. For this, splenocyte cells were washed and suspended in MACS buffer (2-4×10⁶ cells in 600 μL) prior to addition of 2 μL of mouse IgG (ChromPure, Jackson Immunoresearch, cat. No. 015-000-003, at 20 μg/mL), incubating on ice for 15 min. Cells were washed and re-suspended in 40 μL of cold MACS buffer. Non-B cells were magnetically-labeled by addition of 10 μL of Pan B cell Biotin Antibody Cocktail at 4° C., followed by 304, of cold MACS buffer and 20 μL of Anti-Biotin Microbeads. The final volume was adjusted to 5004, with cold MACS buffer.

Non-B cells were removed by passing the cell suspension through an LD column (Miltenyi Biotec, 130-042-901) in a magnetic field using the QuadroMACS™ Separator (Miltenyi Biotec, 130-091-051), collecting flow through.

In parallel, hROR2-TwinStrep (12 μg, from Example 1) was incubated with Strep-Tactin® Magnetic Nanobeads (IBA 6-5500-005, 50 μL at 7.044 mg/mL) and MACS buffer at 4° C., and loaded onto an LS column (Miltenyi Biotec, 130-042-401) in a magnetic field to wash off unbound antigen. Antigen-loaded beads were then resuspended with the LD-column flow through (containing the B cells) and incubated 45 min on ice. After centrifugation and washing with MACS buffer, the mixture was placed in an LS column, washing under a magnetic field. Cells subsequently washed off the beads outside of the magnetic field are antigen-positive B cells.

Antibody cDNA Library Generation.

Antigen-positive B cells were re-suspended in 5004, TRI Reagent; 1004, of chloroform was added following 5 min incubation. After vortex application and centrifugation, the clear upper phase was transferred to an Eppendorf tube, to which 1 μL of glycogen and 2504, isopropanol were added. Following mixing, incubation, washing and centrifugation, supernatant was removed by decanting and 5004, of ice-cold ethanol (75%) were added to the RNA pellet, mixing by inverting. The RNA pellet was then centrifuged and the supernatant decanted before air-drying.

RNA was reverse transcribed to cDNA using standard techniques. Variable domains were amplified in a PCR cycler with human primers in two steps: a first set of primers, as per Table 6, (a) complemented the 5′ end of the variable domain, with inclusion of an upstream nucleotide sequence corresponding to part of a leader peptide, and (b) complemented the 5′ end of the rat constant domain. A second set of primers included: (a) to complement the 5′ end, nucleotide sequences allowing production of the full leader peptide, as well as the NotI restriction site; (b) to amplify the fully-human variable domain relative to the 3′ end, using nucleotide sequence binding to the very 3′end of the J-domain, as well as NheI or BsiWI restriction sites for IgG and IgM, or IgK, respectively.

TABLE 6 Primers used for cDNA library generation. Primer name Sequence Reference 1^(st) step PCR IgG and IgM forward primer set huVH4B-3′Leader-FR1 CAGGTGCAGCTGCAGGAGTCSG Sblattero and huVH5B-3′Leader-FR1 CAGGTACAGCTGCAGCAGTCA Bradbury 1998 huVH6B-3′Leader-FR1 CAGGTGCAGCTACAGCAGTGGG huVH10Ba-3′Leader- GAGGTGCAGCTGKTGGAGWCT FR1 huVH10Bb-3′Leader- GAGGTGCAGCTGKTGGAGWCC FR1 huVH12B-3′Leader-FR1 CAGGTCCAGCTKGTRCAGTCTGG huVH14B-3′Leader-FR1 CAGRTCACCTTGAAGGAGTCTG huVH22B-3′Leader-FR1 CAGGTGCAGCTGGTGSARTCTGG 1^(st) step PCR IgG reverse primer rat_IgG12abc_R CAGGGTGACTGAGGGCGTAG developed in house 1^(st) step PCR IgM reverse primer rat_IgM_R GTTGGGAAGGTTCTGACACC developed in house 1^(st) step PCR IgK forward primer set hu5′VK1-5_3′Leader- GACATCCAGATGACCCAGTC Tiller et al. 2008 FR1 hu5′VK1-9_3′Leader- GACATCCAGTTGACCCAGTCT FR1 hu5′VK1D-43_3′Leader- GCCATCCGGATGACCCAGTC FR1 hu5′VK2-24_3′Leader- GATATTGTGATGACCCAGAC FR1 hu5′VK2-28_3′Leader- GATATTGTGATGACTCAGTC FR1 hu5′VK2-30_3′Leader- GATGTTGTGATGACTCAGTC FR1 hu5′VK3-11_3′Leader- GAAATTGTGTTGACACAGTC FR1 hu5′VK3-15_3′eader- GAAATAGTGATGACGCAGTC FR1 hu5′VK3-20_3′Leader- GAAATTGTGTTGACGCAGTCT FR1 hu5′VK4-1_3′leader- GACATCGTGATGACCCAGTC FR1 1^(st) step PCR IgK reverse primer rat_CK_R CTTGACACTGATGTCTCTGGG developed in house 2^(nd) step PCR IgG and IgM reverse primer set hu3JH1245_NheI TGAGGAGACGGTGACCAG Tiller et al. 2008 hu3LJH3_NheI TGAAGAGACGGTGACCATTG hu3LJH6_NheI TGAGGAGACGGTGACCGTG 2^(nd) step PCR IgK reverse primer set huHSCJK2o-B_BsiWI TTTGATCTCCAGCTTGGTCCC Berry et al., 2003 huHSCJK3o-B_BsiWI TTTGATATCCACTTTGGTCCC huHSCJK5o-B_BsiWI TTTAATCTCCAGTCGTGTCCC huHSCJK14o-B_BsiWI TTTGATYTCCACCTTGGTCCC

Transposition-Mediated Generation of Cellular Libraries and Library Characterization.

Antibody variable-domain encoding cDNA's were then cloned into vectors via the encoded restriction sites alongside nucleotides encoding the human constant domains, and between nucleotides encoding sequences functional with PiggyBac transposase, as well as ampicillin resistance. The library was characterized by Miniprep followed by sequencing. The library consisted of 6×10⁶ to 5×10⁷ members.

These were co-transfected into 63-12 murine A-MuLV transformed preB-cells with a second vector encoding the PiggyBac transposase for transposition-mediated B-cell surface display and secretion of antibodies (“Transpo-mAb Display”), as described in Waldmeier et al., 2016.

Functional Screening of Clone Supernatants and Clone Selection.

B cells were single-cell FACS sorted with a FACSAria, and selected based on double-positive staining with an anti-Strep tag antibody (IBA, 2-1555-050) and a polyclonal anti-IgG-PE (ebioscience, 12-4998-82); up to 288 clones per IgG/IgK and IgM/IgK library per mouse were selected and grown in SF-IMDM Medium with 2% (v/v) Fetal Calf Serum (FCS), 100 IU/mL of Pen-Strep-Fungizone, 50 μM Beta-Mercaptoethanol and 2 mM L-glutamine (all Bioconcept, Allschwil, Switzerland)) at 37° C. and 7.5% CO₂ (FIG. 3). Antibodies in selected B-cell clone supernatants were then evaluated by two means: (1) for hROR2 binding by ELISA, and (2) for mediating killing of hROR2-overexpressing EMT-6 cells following hROR2 binding, using a secondary ADC binding to anti-ROR2 antibodies.

hROR2 Binding by One-Spot ELISA

Half of the wells of 96-well plates were coated with 504, of 2 μg/mL of anti-human Fc (Jackson Immunoresearch, 109-006-008), and the other half with 50 μL of 2 μg/mL of hROR2-Twin-Strep (from Example 1), both in coating buffer (0.1M Na₂CO₃, 0.1M NaHCO₃, pH 9.6), and stored overnight at 4° C. After blocking each well with addition of 150 μL of PBS supplemented with 0.05% (v/v) of Tween 20 and 3% (w/v) bovine serum albumin (BSA) for 1 h at 37° C., 50 μL of diluted supernatants (diluted 5-fold in PBS supplemented with 1% (w/v) bovine serum albumin (BSA) and 0.05% (v/v) of Tween 20) were added to each well. For comparison, a serial 1:20 dilution of a purified anti-ROR2-antibody as well as an isotype control antibody starting at 0.5 μg/ml in PBS supplemented with 1% (w/v) bovine serum albumin (BSA) and 0.05% (v/v) of Tween 20 was added per plate. Following 1 h incubation at 37° C., plates were washed with PBS supplemented with 0.05% (v/v) of Tween 20 before addition of HRP-conjugated anti-human IgG (Jackson Immunoresearch, 109-036-008). Following further plate washing, plates were developed with 50 μL of Sigmafast OPD Tablet set (Sigma, P9187) and the reaction stopped by addition of 2M sulfuric acid. Plates were read on an ELISA plate reader (OD 490).

One-Spot Secondary Killing Assay:

EMT6 cells engineered to overexpress hROR2 of Example 7 were plated at 1000 cells per 100 μl per well in DMEM complete (Dulbecco's Modified Eagle Medium (DMEM) High Glucose (4.5 g/L) with L-Glutamine with 10% (v/v) Fetal Calf Serum (FCS), 100 IU/mL of Pen-Strep-Fungizone and 2 mM L-glutamine (all Bioconcept, Allschwil, Switzerland)) and incubated at 37° C. and 5% CO₂. The following day, 15 μl undiluted supernatant or 15 μl of a 2-fold serial dilution of a purified anti-ROR2 antibody starting at 10 μg/ml were added to the cells. Following incubation for 30 min, 35 μl of anti-hu-IgG-CL-PNU (Moradec, AH-102PN-50) was added at 0.32 μg/ml. After an additional three days, plates were removed from the incubator and equilibrated to room temperature. After approximately 30 min, 504 was removed from each well, and then 50 μL of CellTiterGlo® 2.0 Luminescent Solution (Promega, G9243) was added to each well. After shaking the plates at 750 rpm for 5 min followed by 20 min incubation without shaking, luminescence was measured on a Spark 10M plate reader with an integration time of 1 second per well.

Antibodies showing high ROR2-binding as well as low cell viability, thereby indicating being a good ADC candidate, were further analyzed in titrated ELISA and cell viability assays. In the titrated ELISA, the same set-up as described above in the one-spot ELISA was used, with the exception that the 5-fold diluted supernatant was serially diluted 3-fold for 8 wells total. In the titrated secondary killing assay, a similar set-up as described above was used. Here, IgG concentrations of all supernatants were adjusted to the IgG concentration of the lowest expressor (as measured by the titrated ELISA when coating with anti-IgG). From this concentration, all supernatants were serially diluted 2-fold for 6 dilutions total and thereof 15 μl were added to the plate. Analysis was performed using Graphpad Prism Software.

Example 2: Anti-ROR2 Antibody Expression and Purification

Expression Vectors:

Antibody sequences determined above to bind hROR2 were synthesized as DNA by GenScript (Piscataway, USA) and included within an expression vector containing suitable restriction sites and the appropriate constant domain.

Expression and Purification of Anti-ROR2 Antibodies:

Expression vectors were transfected into HEK293T cells using Lipofectamine® LTX Reagent with PLUS™ Reagent (Thermo Fisher Scientific, Reinach, Switzerland, 15388100); following a 1-day incubation (37° C., 5% CO₂, growth media: Dulbecco's Modified Eagle Medium (DMEM) High Glucose (4.5 g/L) with L-Glutamine with 10% (v/v) Fetal Calf Serum (FCS), 100 IU/mL of Pen-Strep-Fungizone and 2 mM L-glutamine (all Bioconcept, Allschwil, Switzerland)), cells were expanded under selection conditions (2 μg/mL of puromycin (Sigma-Aldrich, Buchs SG, Switzerland, P8833-25 mg stock at 2 mg/mL)). Cells were split and further expanded (37° C., 5% CO₂); once confluency was reached, tissue culture dishes were coated with 20 μg/ml poly-L-Lysine (Sigma-Aldrich, P1524) for 2 h at 37° C. and washed twice with PBS. Then, cells were trypsinized and split 1:3 onto poly-L-lysine-coated plates. Again after reaching confluency, cells were washed with PBS followed by media replacement to production media (DMEM/F-12, Gibco/Thermo Fisher Scientific, 31330-03) supplemented with 1 μg/mL puromycin (Sigma, P8833), 100 IU/mL of Pen-Strep-Fungizone (Bioconcept), 161 μg/mL of N-acetyl-L-cysteine (Sigma-Aldrich, A8199) and 10 μg/mL of L-glutathione reduced (Sigma-Aldrich, G6529). Supernatant, harvested bi-weekly and filtered (0.22 μm) to remove cells, was stored at 4° C. until purification.

For purification, filtered supernatant was loaded onto a PBS-equilibrated Protein A HiTrap column (GE Healthcare, Frankfurt am Main, Germany, 17-0405-01) or a JSR Amsphere™ Protein A column (JSR Life Sciences, Leuven, Belgium, JWT203CE) and washed with PBS; elution was performed using 0.1M glycine (pH 2.5) on an AEKTA pure (GE Healthcare). Fractions were immediately neutralized with 1M Tris-HCl buffer (pH 8.0), and analyzed for protein purity and integrity by SDS-PAGE. Protein-containing fractions were mixed and subjected to buffer exchange using Amicon filtration units (Millipore, Schaffhausen, Switzerland, UFC901008) to reach a dilution of 1:100, and then sterile filtered using a low retention filter (0.20 μm, Carl Roth, Karlsruhe, Germany, Pa.49.1).

The purity and integrity of the recombinant antibodies was analyzed by SDS-PAGE.

TABLE 7 Sequences of prior art anti-hROR1 isotype control antibody XBR1-402 and anti-CD30 isotype control antibody Ac10, with the constant domain underlined SEQ ID NO. Amino Acid Sequence (with constant domain underlined) SEQ ID NO. 131 QEQQKESGGGLFKPTDTLTLTCTASGFDISSYYMSWVRQAPGNGLEWIGAIGI XBR1-402 HC SGNAYYASWAKSRSTITRNTNLNTVTLKMTSLTAADTATYFCARDHPTYGMDL amino acid WGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN sequence SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 132 SYELTQLPSVSVSLGQTARITCEGNNIGSKAVHWYQQKPGLAPGLLIYDDDER XBR1-402 LC PSGVPDRFSGSNSGDTATLTISGAQAGDEADYYCQVWDSSAYVFGGGTQLTVT amino acid GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGV sequence ETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO. 133 QIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYP Ac10 HC amino GSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFAY acid sequence WGQGTQVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 134 DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKVLTY Ac10 LC amino AASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGT acid sequence KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

Table 8 lists the protocols used for expression and purification of antibody batches used in the subsequent examples, along with their final concentration and buffer.

TABLE 8 Protocols and concentrations of mAbs used in the Examples C-Terminal Tags Final Antibody Antibody SEQ ID (HC: Heavy Chain, LC: concentration (ref.) HC/LC Light Chain) Buffer (mg/mL) GK-1E5 HC: SEQ ID NO. 6 HC: LPETG-Strep PBS 1.27 (mAb270) LC: SEQ ID NO. 7 LC: G₅SLPETG-Strep GK-5A1 HC: SEQ ID NO. 8 HC: LPETG-Strep PBS 4.5 (mAb271) LC: SEQ ID NO. 9 LC: G₅SLPETG-Strep MK-3B12 HC: SEQ ID NO. 2 HC: LPETG-Strep PBS 3.1 (mAb272) LC: SEQ ID NO. 3 LC: G₅SLPETG-Strep GK-2G8 HC: SEQ ID NO. 10 HC: LPETG-Strep PBS 0.3 (mAb273) LC: SEQ ID NO. 11 LC: G₅SLPETG-Strep GK-6B10 HC: SEQ ID NO. 14 HC: LPETG-Strep PBS 1.6 (mAb279) LC: SEQ ID NO. 15 LC: G₅SLPETG-Strep GK-5E1 HC: SEQ ID NO. 12 HC: LPETG-Strep PBS 2.6 (mAb280) LC: SEQ ID NO. 13 LC: G₅SLPETG-Strep GK-5G12 HC: SEQ ID NO. 16 HC: LPETG-Strep PBS 1.4 (mAb281) LC: SEQ ID NO. 17 LC: G₅SLPETG-Strep MK-7C3 HC: SEQ ID NO. 4 HC: LPETG-Strep PBS 1.3 (mAb282) LC: SEQ ID NO. 5 LC: G₅SLPETG-Strep MK-3B12 HC: SEQ ID NO. 2 HC: LPETG-TwinStrep PBS 3.5 (mAb328) LC: SEQ ID NO. 3 LC: G₅SLPETG-TwinStrep GK-21D3 HC: SEQ ID NO. 18 HC: LPETG-TwinStrep PBS 0.9 (mAb399) LC: SEQ ID NO. 19 LC: G₅SLPETG-TwinStrep GK-21D3 HC: SEQ ID NO. 18 HC: LPETG-TwinStrep PBS 1.8 (mAb417) LC: SEQ ID NO. 19 LC: G₅SLPETG-TwinStrep MK-24C10 HC: SEQ ID NO. 20 HC: LPETG-TwinStrep PBS 6.3 (mAb396) LC: SEQ ID NO. 21 LC: G₅SLPETG-TwinStrep MK-24C10 HC: SEQ ID NO. 20 HC: LPETG-TwinStrep PBS 2.4 (mAb428) LC: SEQ ID NO. 21 LC: G₅LPETG-TwinStrep MK-24F9 HC: SEQ ID NO. 22 HC: LPETG-TwinStrep PBS 4.7 (mAb416) LC: SEQ ID NO. 23 LC: G₅SLPETG-TwinStrep GK-22G12 HC: SEQ ID NO. 24 HC: LPETG-TwinStrep PBS 3.0 (mAb409) LC: SEQ ID NO. 25 LC: G₅SLPETG-TwinStrep GK-22G12 HC: SEQ ID NO. 24 HC: LPETG-TwinStrep PBS 3.0 (mAb418) LC: SEQ ID NO. 25 LC: G₅LPETG-TwinStrep XBR1-402 HC: SEQ ID NO. 71 HC: LPETG-Strep PBS 6.0 (mAb003) LC: SEQ ID NO. 72 LC: G₅SLPETG-Strep Ac10 HC: SEQ ID NO. 73 HC: LPETG-Strep PBS 7.9 (mAb046) LC: SEQ ID NO. 74 LC: G₅SLPETG-Strep

Example 3: Antibody Binding to hROR2 by SPR

Surface plasmon resonance for the measurement of the affinities of anti-hROR2 antibodies to hROR2 was performed on a Biacore T200 instrument (GE Healthcare). Antibodies were captured using a CM5 Protein A sensor chip (GE Healthcare, 29127556) or Protein G was immobilized on a CM5 sensor chip. For affinity measurements, either purified anti-hROR2 antibodies or 293T supernatants containing anti-hROR2 antibodies were used. In all cases, anti-hROR2 antibodies were diluted to 1-3 μg/mL in 1×HBS-EP+ running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA (pH 7.4), and 0.05% (v/v) Tween 20) and captured for 30 s with a flow of 304/min. hROR2-TwinStrep (from Example 1) was diluted in running buffer using 2-fold serial dilutions ranging from 40 nM to 2.5 nM.

Association and dissociation were measured at a flow of 30 μL/min for 120 s and 200 s, respectively. Calculation of association (k_(on)) and dissociation (k_(off)) rate constants was based on a 1:1 Langmuir binding model. The equilibrium dissociation constant (K_(d)) was calculated from k_(off)/k_(on). SPR sensorgrams for the antibodies of Table 9 are shown in FIG. 4, with K_(d), k_(on), and k_(off) values reported in Table 9.

TABLE 9 Binding characteristics of anti-ROR2 antibodies as determined by SPR Antibody k_(on) (M⁻¹s⁻¹) k_(off) (s⁻¹) K_(d) (nM) GK-1E5 (mAb270) 4.83 × 10⁴ 1.23 × 10⁻⁴ 2.5 GK-5A1 (mAb271) 4.32 × 10⁵ 2.65 × 10⁻² 68.4 MK-3B12 (mAb272) 1.21 × 10⁶ 1.02 × 10⁻² 8.4 MK-7C3 (mAb273) 1.53 × 10⁵ 9.16 × 10⁻³ 60.0 GK-2G8 (mAb279) 8.93 × 10⁵ 2.86 × 10⁻² 32.1 GK-6B10 (mAb280) 1.72 × 10⁵ 1.00 × 10⁻³ 5.8 GK-5E1 (mAb281) 1.82 × 10⁵ 1.47 × 10⁻² 80.7 GK-5G12 (mAb282) 1.69 × 10⁵ 1.11 × 10⁻³ 6.6 GK-21D3 (mAb399) 6.55 × 10⁴ 2.28 × 10⁻² 347 MK-24C10 (mAb396) 4.17 × 10⁵ 8.93 × 10⁻³ 21.4 MK-24F9 (mAb416) 3.88 × 10⁵ 4.65 × 10⁻⁴ 1.2 GK-22G12 (mAb409) 6.03 × 10⁵ 4.03 × 10⁻⁴ 0.7

Example 4: Expression of Mouse and Cynomolgus ROR2

Mouse ROR2 (mROR2) and cynomolgus ROR2 (cROR2) were each expressed according to an analogous protocol as given for hROR2 expression in Example 1.

Example 5: Cross-Reactivity of mAbs with Mouse and Cynomolgus ROR2

Binding of antibodies to hROR2, mouse ROR2 (mROR2) and cynomolgus ROR2 (cROR2) was evaluated in an ELISA-based assay. For this, ELISA plates were coated with 50 μL of 2 μg/ml, in sodium bicarbonate coating buffer (0.1M Na₂CO₃, 0.1M NaHCO₃, pH 9.6), of: (a) an anti-human Fc (Jackson Immunoresearch, 109-006-098), (b) hROR2-TwinStrep, (c) cynomolgus ROR2-TwinStrep or (d) mouse ROR2-TwinStrep. Following 1-2-days incubation at 4° C., plates were washed with PBS supplemented with 0.05% (v/v) of Tween 20 and blocked with 150 μL of PBS supplemented with 0.05% (v/v) of Tween 20 and 3% of bovine serum albumin (BSA). Thereafter, 3-fold or 4-fold dilutions of purified antibodies starting at 0.5 or 2 μg/ml, respectively, in PBS supplemented with 1% (w/v) bovine serum albumin (BSA) and 0.05% (v/v) of Tween 20 were added. Following 1 h incubation at 37° C., plates were washed with PBS supplemented with 0.05% (v/v) of Tween 20 prior to addition of HRP-conjugated anti-human IgG (Jackson Immunoresearch, 109-036-008). Following further plate washing, plates were developed with 50 μL of Sigmafast OPD Tablet set (Sigma, P9187) and the reaction stopped by addition of 2M sulfuric acid. Plates were read on an ELISA plate reader (OD 490).

FIG. 5 and FIG. 11 show the ELISA profiles of the mAbs of Table 10. This table also summarizes the binding status of each mAb to ROR2 from each of the evaluated species.

TABLE 10 Results of ELISA-based evaluation of binding of mAbs to human ROR2 (hROR2), cynomolgus ROR2 (cROR2) and mouse ROR2 (mROR2) Antibody hROR2 cROR2 mROR2 GK-1E5 (mAb270) Yes No No GK-5A1 (mAb271) Yes Yes Yes MK-3B12 (mAb272) Yes Yes No MK-7C3 (mAb273) Yes No No GK-2G8 (mAb279) Yes No No GK-6B10 (mAb280) Yes No No GK-5E1 (mAb281) Yes Weak No GK-5G12 (mAb282) Yes No No GK-21D3 (mAb417) Yes Weak No MK-24C10 (mAb428) Yes Yes Weak MK-24F9 (mAb416) Yes Yes Weak GK-22G12 (mAb418) Yes Yes No

Example 6: Conjugation of mAbs with Glycine-Modified Toxins to Form ADCs Using SMAC-Technology™

Sortase A.

Recombinant and affinity purified Sortase A enzyme from Staphylococcus aureus was produced in E. coli as disclosed in WO2014140317A1.

Generation of Glycine-Modified Toxins.

Pentaglycine-modified EDA-anthracycline derivative G5-PNU and triglycine-modified EDA-anthracycline derivative G3-PNU (FIGS. 6 (A) and (B), respectively) were manufactured by Concortis, San Diego, U.S. The identity and the purity of the glycine-modified toxins were confirmed by mass-spectrometry and HPLC. Each of the glycine-modified toxins exhibited >95% purity, as determined by HPLC chromatography.

Sortase-Mediated Antibody Conjugation.

The above-mentioned toxins were conjugated to anti-ROR2 and comparative antibodies as per Table 7 by incubating LPETG-tagged mAbs [5-10 μM] with glycine-modified toxin [100-200 μM] and 2.5-3 μM Sortase A in the listed conjugation buffer for 3.5 h at 25° C. The reaction was stopped by passage through a BioRad Protein A GraviTrap column as per Table 11. Bound conjugate was eluted with 5 column volumes of elution buffer (0.1M glycine pH 2.5, 50 nM NaCl), with 1 column volume fractions collected into tubes containing 25% v/v 1M Tris-Base to neutralise the acid. Protein containing fractions were pooled and formulated in the formulation buffer of Table 11 using a ZebaSpin desalting column.

Adc Analytics.

DAR was assessed by Reverse Phase Chromatography performed on a Polymer Labs PLRP 2.1 mm×5 cm, 5 μm column run at 1 mL/min/80° C. with a 25 min linear gradient of 0.05 to 0.1% TFA/H₂O and 0.04 to 0.1% TFA/CH₃CN. Samples were first reduced by incubation with DTT at pH 8.0 at 37° C. for 15 min. The DAR determined by Reverse Phase Chromatography is summarized in Table 7 below.

TABLE 11 Analytical summary of ADCs manufactured in this study. DAR, drug- to-antibody ratio, determined by reverse phase chromatography. Formulation ADC (ref.) mAb (ref.) Conjugation Buffer Buffer DAR GK-1E5-G5- GK-1E5 50 mM HEPES, pH 7.5, 1 mM CaCl₂, PBS ND PNU (adc442) (mAb270) 10% Glycerol, 150 mM NaCl GK-5A1-G5- GK-5A1 50 mM HEPES, pH 7.5, 1 mM CaCl₂, PBS ND PNU (adc443) (mAb271) 10% Glycerol, 150 mM NaCl MK-3B12-G5- MK-3B12 50 mM HEPES, pH 7.5, 1 mM CaCl₂, PBS ND PNU (adc444) (mAb272) 10% Glycerol, 150 mM NaCl GK-2G8-G5- GK-2G8 50 mM HEPES, pH 7.5, 1 mM CaCl₂, PBS 3.7 PNU (adc445) (mAb273) 10% Glycerol, 150 mM NaCl GK-6B10-G5- GK-6B10 50 mM HEPES, pH 7.5, 1 mM CaCl₂, PBS 3.6 PNU (adc446) (mAb279) 10% Glycerol, 150 mM NaCl GK-5E1-G5- GK-5E1 50 mM HEPES, pH 7.5, 1 mM CaCl₂, PBS 3.6 PNU (adc447) (mAb280) 10% Glycerol, 150 mM NaCl GK-5G12-G5- GK-5G12 50 mM HEPES, pH 7.5, 1 mM CaCl₂, PBS 3.8 PNU (adc448) (mAb281) 10% Glycerol, 150 mM NaCl MK-7C3-G5- MK-7C3 50 mM HEPES, pH 7.5, 1 mM CaCl₂, PBS ND PNU (adc449) (mAb282) 10% Glycerol, 150 mM NaCl MK-3B12-G3- MK-3B12 50 mM HEPES, pH 7.5, 1 mM CaCl₂, PBS 3.9 PNU (adc487) (mAb328) 10% Glycerol, 150 mM NaCl GK-21D3-G5- GK-21D3 50 mM HEPES, pH 7.5, 1 mM CaCl₂, PBS 3.3 PNU (adc701) (mAb399) 10% Glycerol, 150 mM NaCl MK-24C10-G5- MK-24C10 50 mM HEPES, pH 7.5, 1 mM CaCl₂, PBS 2.9 PNU (adc719) (mAb396) 10% Glycerol, 150 mM NaCl MK-24F9-G5- MK-24F9 50 mM HEPES, pH 7.5, 1 mM CaCl₂, PBS 3.7 PNU (adc718) (mAb416) 10% Glycerol, 150 mM NaCl GK-22G12-G5- GK-22G12 50 mM HEPES, pH 7.5, 1 mM CaCl₂, PBS 3.5 PNU (adc717) (mAb409) 10% Glycerol, 150 mM NaCl XBR1-402-G5- XBR1-402 10 mM sodium succinate, pH 5.0, 10 mM sodium 3.6 PNU (adc096) (mAb003) 175 mM sucrose, 0.02% (w/v) succinate, pH 5.0, Tween 20 175 mM sucrose, 0.02% w/v Tween 20 Ac10-G5-PNU Ac10 50 mM HEPES, pH 7.5, 1 mM CaCl₂, PBS 3.7 (adc332) (mAb046) 10% Glycerol, 150 mM NaCl

From these analyses it can be concluded that the SMAC-Technology™ conjugation has proceeded at high efficiency resulting in overall average DARs in the range of ca. 3.5 to 4.0 for each of the anti-hROR2 antibody-toxin combinations.

Example 7: EMT-6 Cell Engineering to Stably Express hROR2

EMT-6 cells cultured in DMEM complete (Dulbecco's Modified Eagle Medium (DMEM) High Glucose (4.5 g/L) with L-Glutamine with 10% (v/v) Fetal Calf Serum (FCS), 100 IU/mL of Pen-Strep-Fungizone and 2 mM L-glutamine (all Bioconcept, Allschwil, Switzerland)) at 37° C. and 5% CO₂ were centrifuged (6 min, 1200 rpm, 4° C.) and suspended in RPMI-1640 media (5×10⁶ cells/mL). 400 μL of this cell suspension was then added to 400 μL of RPMI containing 10.2 μg of the transposable vector pPB-PGK-Puro-hROR2-Thr, directing co-expression of full-length ROR2 and the puromycin-resistance gene, and 3.6 μg of transposase-containing vector pCDNA3.1_hy_mPB. DNA/EMT-6 cell mixture was transferred to electroporation cuvettes (0.4 cm-gap, 165-2088, BioRad, Cressler, Switzerland) and electroporated using the Biorad Gene Pulser II with capacitance extender at 300V and 950 μF. Then, cells were incubated for 5-10 min at room temperature. Following the incubation, cells were centrifuged at 1200 rpm for 6 min, washed once and subsequently resuspended in DMEM complete prior to incubation at 37° C. in a humidified incubator at 5% CO₂ atmosphere. One day after electroporation, cell pools stably expressing hROR2 were selected based on puromycin resistance using 3 μg/mL of puromycin (Sigma-Aldrich, P8833-25 mg stock at 2 mg/mL).

ROR2 expression on selected EMT-6 ROR2 cells was confirmed by flow cytometry. Briefly, following trypsinization, 10⁶ cells were centrifuged in FACS tubes; obtained pellets were resuspended in buffer (PBS with 2% (v/v) FCS). Cells were then incubated with anti-ROR2 mAb Orb38364 (Biorbyt; 30 min, 4° C., final concentration 2 μg/mL), followed by centrifugation and washing. Cells were then resuspended as previously and incubated with a mouse anti-rabbit IgG antibody PE (Abcam, ab99704) with a 1:250 dilution in the dark (30 min, 4° C.), prior to washing.

Using a FACS Aria II, cells were single cell sorted into a 96-well flat-bottom plate containing 200 μL of DMEM complete per well. This plate was incubated at 37° C. and expanded to 6 wells before screening. Cells were analyzed using a FACSCalibur instrument (BD Biosciences) and FlowJo analytical software (Tree Star, Ashland, Oreg.). FIG. 7 shows the FACS analysis data of the high ROR2-expressing clone 14.

Example 8: In Vitro Cytotoxicity Assays of PNU-Based ADCs on hROR2-Expressing EMT-6 Cancer Cells

Cytotoxicity of the anti-ROR2 ADCs of Table 12 were investigated using the EMT-6 engineered cell line of Example 7.

For this, 1000 engineered EMT6 cells (clone 14) per well were plated on 96-well plates (excluding edge wells, which contained water) in 75 μL DMEM supplemented with 10% by vol. FCS, 100 IU/mL Pen-Strep-Fungizone and 2 mM L-Glutamine at a density of 1.3×10⁵ cells per well, and were grown at 37° C. in a humidified incubator at 5% CO₂ atmosphere. After a 1-day incubation, each ADC was added to respective wells in an amount of 25 μL of 3.5-fold serial dilutions in growth medium (starting ADC concentration of 80 μg/mL, giving final ADC concentrations ranging from around 20 μg/ml to 0.3 ng/ml). Each dilution was done in duplicate. After 4 additional days, plates were removed from the incubator and equilibrated to room temperature. After approximately 30 min, 50 μL was removed from each well, and then 50 μL of CellTiter-Glo® 2.0 Luminescent Solution (Promega, G9243) was added to each well. After shaking the plates at 750 rpm for 5 min followed by 20 min incubation without shaking, luminescence was measured on a Spark 10M plate reader with an integration time of 1 second per well. Curves of luminescence versus ADC concentration (ng/mL) were fitted with Graphpad Prism Software. The measurement was repeated twice. The IC₅₀ values, determined using the built-in “log(inhibitor) vs. response—Variable slope (four parameters)” IC₅₀ determination function of Prism Software, are reported in Table 12.

TABLE 12 In vitro cell killing by anti-ROR2 ADCs (ng/mL) ADC/Cell type EMT-6 ROR2 GK-1E5-G5-PNU (adc442) 15.5 GK-5A1-G5-PNU (adc443) 341 MK-3B12-G5-PNU (adc444) 12.9 GK-2G8-G5-PNU (adc445) 15.6 GK-6B10-G5-PNU (adc446) 15.2 GK-5E1-G5-PNU (adc447) 6.5 GK-5G12-G5-PNU (adc448) 15.3 MK-7C3-G5-PNU (adc449) 46.7

FIG. 8 shows the dose-response curve of the in vitro cell killing assays on EMT-6 breast cancer cells stably expressing hROR2 with the PNU-based ADCs of Table 12. As per Table 12 and FIG. 8, ADCs based on the novel anti-ROR2 antibodies of the invention kill ROR2-expressing cells.

Example 9: In Vitro Cytotoxicity Assays of PNU-Based ADCs on hROR2-Expressing EMT-6 Breast Cancer Cells and on ROR2-Negative Cancer Cells

Cytotoxicity of the anti-ROR2 ADCs of Table 13 were investigated using the EMT-6 engineered cell line of Example 7. Low ROR2 expressing human cell line Karpas-299 was used as control. ADC Ac10-G5-PNU was included as isotype control.

For this, 1×10³ engineered EMT6-ROR2 (clone 14) and 2.5×10³ Karpas-299 cells, per well, were each plated on 96-well plates (excluding edge wells, which contained water) in 75 μL DMEM supplemented with 10% by vol. FCS, 100 IU/mL Pen-Strep-Fungizone and 2 mM L-Glutamine at a density of 1.3×10⁵ cells per well, and were grown at 37° C. in a humidified incubator at 5% CO₂ atmosphere. After a 1-day incubation, each ADC was added to respective wells in an amount of 25 μL of 3.5-fold serial dilutions in growth medium (starting ADC concentration of 80 μg/mL, giving final ADC concentrations ranging from 20 μg/ml to 0.89 ng/ml). Each dilution was done in duplicate. After 4 additional days, plates were removed from the incubator and equilibrated to room temperature. After approximately 30 min, 50 μL was removed from each well, and then 50 μL of CellTiter-Glo® 2.0 Luminescent Solution (Promega, G9243) was added to each well. After shaking the plates at 750 rpm for 5 min followed by 20 min incubation without shaking, luminescence was measured on a Spark 10M plate reader with an integration time of 1 second per well. Curves of luminescence versus ADC concentration (ng/mL) were fitted with Graphpad Prism Software. The IC₅₀ values, determined using the built-in “log(inhibitor) vs. response—Variable slope (four parameters)” IC₅₀ determination function of Prism Software, are reported in Table 13.

TABLE 13 In vitro cell killing by anti-ROR2 ADCs (ng/mL) EMT-6 ROR2 Karpas-299 ADC/Cell type clone 14 Low ROR2- hROR2 expression status Positive positive MK-3B12-G5-PNU (adc444) 50.5  3′930 MK-3B12-G3-PNU (adc487) 6.4   565 AC10-G5-PNU (adc332) 5′663    1.8 × 10⁵

Example 10: In Vitro Cytotoxicity Assays of PNU-Based ADCs on hROR2-Expressing EMT-6 Cancer Cells

Cytotoxicity of the anti-ROR2 ADCs of Table 14 were investigated using the EMT-6 engineered cell line of Example 7.

For this, 1×10³ engineered EMT6-ROR2 (clone 14), per well, were each plated on 96-well plates (excluding edge wells, which contained water) in 75 μL DMEM supplemented with 10% by vol. FCS, 100 IU/mL Pen-Strep-Fungizone and 2 mM L-Glutamine at a density of 1.3×10⁵ cells per well, and were grown at 37° C. in a humidified incubator at 5% CO₂ atmosphere. After a 1-day incubation, each ADC was added to respective wells in an amount of 25 μL of 3.5-fold serial dilutions in growth medium (starting ADC concentration of 80 μg/mL, giving final ADC concentrations ranging from 20 μg/ml to 0.89 ng/ml). Each dilution was done in duplicate. After 4 additional days, plates were removed from the incubator and equilibrated to room temperature. After approximately 30 min, 50 μL of CellTiter-Glo® 2.0 Luminescent Solution (Promega, G9243) was added to each well. After shaking the plates at 750 rpm for 5 min followed by 20 min incubation without shaking, luminescence was measured on a Spark 10M plate reader with an integration time of 1 second per well. Curves of luminescence versus ADC concentration (ng/mL) were fitted with Graphpad Prism Software. The IC₅₀ values, determined using the built-in “log(inhibitor) vs. response—Variable slope (four parameters)” IC₅₀ determination function of Prism Software, are reported in Table 14.

TABLE 14 In vitro cell killing by anti-ROR2 ADCs (ng/mL) ADC/Cell type EMT-6 ROR2 GK-21D3-G5-PNU (adc701) 36.4 MK-24C10-G5-PNU (adc719) 58.1 MK-24F9-G5-PNU (adc718) 20.9 GK-22G12-G5-PNU (adc717) 22.9

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

All publications, databases, GenBank sequences, patents, and patent applications cited in this specification are herein incorporated by reference as if each was specifically and individually indicated to be incorporated by reference.

SEQUENCES

A WIPO ST 25 compatible electronic sequence listing is provided with this application, too. For the avoidance of doubt, if discrepancies exist between the sequences in the following table and the electronic sequence listing, the sequences in this table shall be deemed to be the correct ones.

No Qualifier Sequence 1 extracellular EVEVLDPNDPLGPLDGQDGPIPTLKGYFLNFLEPVNNITIVQGQTAILHCKV domain of AGNPPPNVRWLKNDAPVVQEPRRIIIRKTEYGSRLRIQDLDTTDTGYYQCV human ROR2 ATNGMKTITATGVLFVRLGPTHSPNHNFQDDYHEDGFCQPYRGIACARFIG NRTIYVDSLQMQGEIENRITAAFTMIGTSTHLSDQCSQFAIPSFCHFVFPLCD ARSRTPKPRELCRDECEVLESDLCRQEYTIARSNPLILMRLQLPKCEALPMPE SPDAANCMRIGIPAERLGRYHQCYNGSGMDYRGTASTTKSGHQCQPWAL QHPHSHHLSSTDFPELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCD VPSCSPRDSSKMG 2 MK-3B12 HC EVQLVESGPGLLKPSETLSLTCAVSGYSISSGYYWGWIRQPPGKGLEWIGSIY QSGSTHYNPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCAREDRAGWY PFDCWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGR 3 MK-3B12 LC DIVMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKAS SLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNNYWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 4 MK-7C3 HC EVQLLETGGGVVQPGRSLRLSCVASGFTFRSHGMHWVRQAPGKGLEWVA LIWYDGSKKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVG AGLYLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 5 MK-7C3 LC AIRMTQSPSTLSASVGDRVTITCRASQTISNWLAWFQQKPGKAPKVLIYKAS SLESGVPSRFSGSGSGTEFTLTISSLQPDDFASYYCQQYNSYSYTFGQGTRLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 6 GK-1E5 HC EVQLVESGGGVVQPGRSLRLSCAASGFTFRSYGMHWVRQAPGKGLEWVA IIWYDGSKKYYTDSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPGI AMTGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 7 GK-1E5 LC DIQLTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASS LESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNNYWTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 8 GK-5A1 HC QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVA VIWNDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREG SGWYDYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9 GK-5A1 LC EIVLTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASS LESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYWTFGQGTKVDI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 10 GK-2G8 HC QVQLQESGGGVVQPGRSLRLSCAASGFTFRSYGMHWVRQAPGKGLEWV AIIWYDGSKKYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPG VAMTGLDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 11 GK-2G8 LC EIVMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKAS SLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNNYWTFGQGTKV DIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 12 GK-5E1 HC QVTLKESGGDVVQPGRSLRLSCAASGFTFRTYGMHWVRQAPGKGPEWVA LIWYDGSKKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRVRF GELYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 13 GK-5E1 LC DIVMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKAS SLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYSFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 14 GK-6B10 HC QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWV ALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARV AAALHFHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 15 GK-6B10 LC DIVMTQSPSTLSASVGDRVTITCRASQSIDNWLAWYQQKPGKAPKVLIYKA SSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 16 GK-5G12 HC QITLKESGGGVVQPGRSLRLSCAASGFTFRTYGMHWVRQAPGKGLEWVA LIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCIRVKF GDLYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 17 GK-5G12 LC EIVLTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAAST LQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPYTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 18 GK-21D3 HC QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWV AIIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRDEDTAVYYCARM GAINRGGGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 19 GK-21D3 LC DIQLTQSPSSLSASIGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTL QSGVPSRFSGSGSGTDFTLTISSLQPEDVSTYYCQKYNSAPWTFGQGTKVDI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 20 MK-24C10 HC QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWV AVIWFDGTNKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DKGEWFGELRYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 21 MK-24C10 LC EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIY LGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQ GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC 22 MK-24F9 HC EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGD INHSRTTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGEQW LVPFDYWDQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 23 MK-24F9 LC EIVMTQSPSTLSASVGDRVTITCRASQSISHWLAWYQQKPGKAPKLLIYKAS SLKSGVPSRFNGSGSGTEFTLTISSLQPDDFATYYCQHYNTYSRTFGQGTKV DIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 24 GK-22G12 HC EVQLVESGGGLVQSGGSLRLSCAASGFTFSSQRLSWVRQAPGKGLEWVAN IKQDGSEKNYVDSVRGRFTISRDIAKNSLYLQMNSLRAEDTAVYYCARDGYR NGWHIPEDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 25 GK-22G12 LC DIVMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAAS TLQSGVPSRFSGSGSGTDFTLTISSLQPEDVSTYYCQKHNRAPWTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 26 MK-3612 HC CDR1 GYSISSGYY 27 MK-3612 HC CDR2 IYQSGST 28 MK-3612 HC CDR3 CAREDRAGWYPFDCW 29 MK-3612 LC CDR1; GK-1E5 LC CDR1; GK-5A1 LC CDR1; QSISSW GK-2G8 LC CDR1; GK-5E1 LC CDR1; GK-1H2 LC CDR1; GK- 2A9 LC CDR1; GK-5A6 LC CDR1 30 MK-3612 LC CDR3; GK-1E5 LC CDR3; GK-2G8 LC CDR3; CQQYNNYWTF GK-1H2 LC CDR3; GK-2A9 LC CDR3 31 MK-7C3 HC CDR1 GFTFRSHG 32 MK-7C3 HC CDR2; GK-1E5 HC CDR2; GK-2G8 HC CDR2; IWYDGSKK GK-5E1 HC CDR2; GK-1H2 HC CDR2; GK-2A9 HC CDR2 33 MK-7C3 HC CDR3 CARVGAGLYLDYW 34 MK-7C3 LC CDR1 QTISNW 35 MK-7C3 LC CDR3; GK-6610 LC CDR3; GK-5A6 LC CDR3 CQQYNSYSYTF 36 GK-1E5 HC CDR1; GK-2G8 HC CDR1; GK-1H2 HC CDR1; GFTFRSYG GK-2A9 HC CDR1 37 GK-1E5 HC CDR3; GK-1H2 HC CDR3 CARPGIAMTGLDYW 38 GK-5A1 HC CDR1; GK-21D3 HC CDR1; MK-24C10 HC GFTFSSYG CDR1; GK-21F1 HC CDR1; MK-24C12 HC CDR1 39 GK-5A1 HC CDR2 IWNDGSNK 40 GK-5A1 HC CDR3 CAREGSGWYDYYYGMDVW 41 GK-5A1 LC CDR3 CQQYNSYWTF 42 GK-5E1 LC CDR3 CQQYNSYSYSF 43 GK-2G8 HC CDR3; GK-5E1 HC CDR1; GK-5G12 HC CDR1; CARPGVAMTGLDLW GK-2A9 HC CDR3; GK-5A6 HC CDR1 44 GK-5E1 HC CDR3 CVRVRFGELYFQHW 45 GK-6B10 HC CDR1 GFTFSRYG 46 GK-6B10 HC CDR2; GK-5G12 HC CDR2; GK-21D3 HC IWYDGSNK CDR2; GK-5A6 HC CDR2; GK-21F1 HC CDR2 47 GK-6B10 HC CDR3 CARVAAALHFHYW 48 GK-6B10 LC CDR1 QSIDNW 49 GK-5G12 HC CDR3; GK-5A6 HC CDR3 CIRVKFGDLYFQHW 50 GK-5G12 LC CDR1; GK-21D3 LC CDR1; GK-22G12 LC QGISNY CDR1; GK-21F1 LC CDR1; GK-21G5 LC CDR1; GK-21E6 LC CDR1; GK-22E12 LC CDR1 51 GK-5G12 LC CDR3 CQKYNSAPYTF 52 GK-21D3 HC CDR3; GK-21F1 HC CDR3 CARMGAINRGGGGFDYW 53 GK-21D3 LC CDR3; GK-21F1 LC CDR3 CQKYNSAPWTF 54 MK-24C10 HC CDR2; MK-24C12 HC CDR2 IWFDGTNK 55 MK-24C10 HC CDR3; MK-24C12 HC CDR3 CARDKGEWFGELRYYYYGM DVW 56 MK-24C10 LC CDR1; MK-24C12 LC CDR1 QSLLHSNGYNY 57 MK-24C10 LC CDR3; MK-24C12 LC CDR3 CMQALQTPYTF 58 MK-24F9 HC CDR1; GK-21G5 HC CDR1; GK-23A8 HC GGSFSGYY CDR1 59 MK-24F9 HC CDR2  INHSRTT 60 MK-24F9 HC CDR3  CARGGEQWLVPFDYW 61 MK-24F9 LC CDR1; GK-23A8 LC CDR1 QSISHW 62 MK-24F9 LC CDR3; GK-23A8 LC CDR3 CQHYNTYSRTF 63 GK-22G12 HC CDR1; GK-21E6 HC CDR1; GK-22E12 HC GFTFSSQR CDR1 64 GK-22G12 HC CDR2; GK-21E6 HC CDR2; GK-22E12 HC IKQDGSEK CDR2 65 GK-22G12 HC CDR3; GK-21E6 HC CDR3; GK-22E12 HC CARDGYRNGWHIPEDYW CDR3 66 GK-22G12 LC CDR3; GK-21G5 LC CDR3; GK-21E6 LC CQKHNRAPWTF CDR3; GK-22E12 LC CDR3 67 CDR1; X₁ = F or G, X₂ = T or S, X₃ = R or S, X₄ = S, T, R or G, GX₁X₂FX₃X₄X₅X₆ X₅ = H, Y or Q, X₆ = G, Y or R 68 CDR2; X₇ = W or K, X₈ = Y, N, F or Q, X₉ = S or T, X₁₀ = K, N or IX₇X₈DGX₉X₁₀K E 69 CDR1; X₁₁ = S, T or G, X₁₂ = S, N or H and X₁₃ = W or Y QX₁₁ISX₁₂X₁₃ 70 CDR3; X₁₄ = Q or M, X₁₅ = K, H or Q, X₁₆ = H, Y or A, X₁₇ = N CX₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂F or L, X₁₈ = R, T, Q, S or N; X₁₉ = A, Y or T; X₂₀ = P, S or W, X₂₁ = W, R, Y or absent, X₂₂ = S or T. 71 CDR1; X₃ = R or S, and X₄ = S, T GFTFX₃X₄YG 72 GK-5G12 LC CDR2; GK-21D3 LC CDR2; GK-22G12 LC AAS CDR2; GK-21F1 LC CDR2; GK-21G5 LC CDR2; GK-21E6 LC CDR2; GK-22E12 LC CDR2 73 MK-3612 LC CDR2; MK-7C3 LC CDR2; GK-1E5 LC CDR2; KAS GK-5A1 LC CDR2; GK-2G8 LC CDR2; GK-5E1 LC CDR2; GK- 6B10 LC CDR2; MK-24F9 LC CDR2; GK-1H2 LC CDR2; GK- 2A9 LC CDR2; GK-5A6 LC CDR2; GK-23A8 LC CDR2 74 MK-24C10 LC CDR2, MK-24C12 LC CDR2 LGS 75 GK-1H2 HC EVQLLESGGGVVQPGRSLRLSCAASGFTFRSYGMHWVRQAPGKGLEWVA IIWYDGSKKYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPGI AMTGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 76 GK-1H2 LC EIVMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKAS SLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNNYWTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 77 GK-2A9 HC QVQLVQSGGGVVQPGRSLRLSCAASGFTFRSYGMHWVRQAPGKGLEWV AIIWYDGSKKYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPG VAMTGLDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 78 GK-2A9 LC EIVLTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASS LESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNNYWTFGQGTKVDI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 79 GK-5A6 HC EVQLQESGGGVVQPGRSLRLSCAASGFTFRTYGMHWVRQAPGKGLEWV ALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCIRVK FGDLYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 80 GK-5A6 LC DVVMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKA SSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 81 G K-21 F1 HC QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWV AIIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRDEDTAVYYCARM GAINRGGGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 82 GK-21F1 LC DIQLTQSPSSLSASIGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTL QSGVPSRFSGSGSGTDFTLTISSLQPEDVSTYYCQKYNSAPWTFGQGTKVDI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 83 M K-24C12 HC EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVA VIWFDGTNKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDK GEWFGELRYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 84 MK-24C12 LC EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIY LGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQ GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC 85 GK-21G5 HC QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGE INHSGITNYNPSLKSRLTVSVDTSKNQFSLKLSSVTAADTAVYYCARGGDQW LVPFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 86 GK-21G5 LC DIVMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAAS TLQSGVPSRFSGSGSGTDFTLTISSLQPEDVSTYYCQKHNRAPWTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 87 GK-23A8 HC QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGE INHSGITNYNPSLKSRLTVSVDTSKNQFSLKLSSVTAADTAVYYCARGGDQW LVPFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 88 GK-23A8 LC EIVMTQSPSTLSASVGDRVTITCRASQSISHWLAWYQQKPGKAPKLLIYKAS SLKSGVPSRFNGSGSGTEFTLTISSLQPDDFATYYCQHYNTYSRTFGQGTKV DIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 89 GK-21E6 HC RVQLVQSGGGLVQSGGSLRLSCAASGFTFSSQRLSWVRQAPGKGLEWVA NIKQDGSEKNYVDSVRGRFTISRDIAKNSLYLQMNSLRAEDTAVYYCARDG YRNGWHIPEDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 90 GK-21E6 LC DVVMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAAS TLQSGVPSRFSGSGSGTDFTLTISSLQPEDVSTYYCQKHNRAPWTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 91 GK-22E12 HC EVQLLESGGGLVQSGGSLRLSCAASGFTFSSQRLSWVRQAPGKGLEWVANI KQDGSEKNYVDSVRGRFTISRDIAKNSLYLQMNSLRAEDTAVYYCARDGYR NGWHIPEDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 92 GK-22E12 LC AIRMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAAS TLQSGVPSRFSGSGSGTDFTLTISSLQPEDVSTYYCQKHNRAPWTFGQGTK VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 93 GK-21G5 HC CDR2, GK-23A8 HC CDR2 INHSGIT 94 GK-5E1 HC CDR1; GK-5G12 HC CDR1; GK-5A6 HC CDR1 GFTFRTYG 95 GK-21G5 HC CDR3, GK-23A8 HC CDR3 CARGGDQWLVPFDNW 96 Staphylococcus aureus sortase A recognition sequence, -LPXSG with X being any amino acid 97 Staphylococcus aureus sortase A recognition sequence, -LPXAG with X being any amino acid 98 recognition sequence for Staphylococcus aureus sortase -LPXTG A or engineered sortase A 4S-9 from Staphylococcus aureus, with X being any amino acid 99 recognition sequence for engineered sortase A 2A-9 from -LAXTG Staphylococcus aureus, with X being any amino acid 100 recognition sequence for engineered sortase A 2A-9 from -LAETG Staphylococcus aureus 101 Streptococcus pyogenes sortase A recognition sequence, -LPXTA with X being any amino acid 102 Staphylococcus aureus sortase recognition sequence -NPQTN 103 huVH4B-3′Leader-FR1 CAGGTGCAGCTGCAGGAGTCSG 104 huVH5B-3′Leader-FR1 CAGGTACAGCTGCAGCAGTCA 105 huVH6B-3′Leader-FR1 CAGGTGCAGCTACAGCAGTGGG 106 huVH10Ba-3′Leader-FR1 GAGGTGCAGCTGKTGGAGWCT 107 huVH10Bb-3′Leader-FR1 GAGGTGCAGCTGKTGGAGWCC 108 huVH12B-3′Leader-FR1 CAGGTCCAGCTKGTRCAGTCTGG 109 huVH14B-3′Leader-FR1 CAGRTCACCTTGAAGGAGTCTG 110 huVH22B-3′Leader-FR1 CAGGTGCAGCTGGTGSARTCTGG 111 rat_IgG12abc_R CAGGGTGACTGAGGGCGTAG 112 rat_IgM_R GTTGGGAAGGTTCTGACACC 113 hu5′VK1-5_3′Leader-FR1 GACATCCAGATGACCCAGTC 114 hu5′VK1-9_3′Leader-FR1 GACATCCAGTTGACCCAGTCT 115 hu5′VK1D-43_3′Leader-FR1 GCCATCCGGATGACCCAGTC 116 hu5′VK2-24_3′Leader-FR1 GATATTGTGATGACCCAGAC 117 hu5′VK2-28_3′Leader-FR1 GATATTGTGATGACTCAGTC 118 hu5′VK2-30_3′Leader-FR1 GATGTTGTGATGACTCAGTC 119 hu5′VK3-11_3′Leader-FR1 GAAATTGTGTTGACACAGTC 120 hu5′VK3-15_3′Leader-FR1 GAAATAGTGATGACGCAGTC 121 hu5′VK3-20_3′Leader-FR1 GAAATTGTGTTGACGCAGTCT 122 hu5′VK4-1_3′Leader-FR1 GACATCGTGATGACCCAGTC 123 rat_CK_R CTTGACACTGATGTCTCTGGG 124 hu3′JH1245_NheI TGAGGAGACGGTGACCAG 125 hu3′JH3_NheI TGAAGAGACGGTGACCATTG 126 hu3′JH6_NheI TGAGGAGACGGTGACCGTG 127 huHSCJK2o-B_BsiWI TTTGATCTCCAGCTTGGTCCC 128 huHSCJK3o-B_BsiWI TTTGATATCCACTTTGGTCCC 129 huHSCJK5 o-B_BsiWI TTTAATCTCCAGTCGTGTCCC 130 huHSCJK14o-B_BsiWI TTTGATYTCCACCTTGGTCCC 131 XBR1-402 LC SYELTQLPSVSVSLGQTARITCEGNNIGSKAVHWYQQKPGLAPGLLIYDDDE RPSGVPDRFSGSNSGDTATLTISGAQAGDEADYYCQVWDSSAYVFGGGTQ LTVTGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKT VAPTECS 132 XBR1-402 HC QEQQKESGGGLFKPTDTLTLTCTASGFDISSYYMSWVRQAPGNGLEWIGAI GISGNAYYASWAKSRSTITRNTNLNTVTLKMTSLTAADTATYFCARDHPTY GMDLWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 133 Ac10 LC QIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWI YPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNY WFAYWGQGTQVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 134 Ac10 LC DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKV LIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTF GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC 135 Linker derived from Staphylococcus aureus sortase A -LPXT(Gn)- recognition sequence, with X being any amino acid and n ≥ 1 and ≤ 21 136 Linker derived from Staphylococcus aureus sortase A -LPXA(Gn)- recognition sequence, with X being any amino acid and n ≥ 1 and ≤ 21 137 Linker derived from recognition sequence for -LPXS(Gn)- Staphylococcus aureus sortase A or engineered sortase A 4S-9 from Staphylococcus aureus, with X being any amino acid and n ≥ 1 and ≤ 21 138 Linker derived from recognition sequence for engineered -LAXT(Gn)- sortase A 2A-9 from Staphylococcus aureus, with X being any amino acid and n ≥ 1 and ≤ 21 139 Linker derived from Streptococcus pyogenes sortase A -LPXT(Gn)- or -LPXT(An)- recognition sequence, with X being any amino acid and n ≥ 1 and =  21 140 Linker derived from Staphylococcus aureus sortase -NPQT(Gn)- recognition sequence, with n ≥ 1 and ≤ 21 141 extracellular domain of cynomolgus ROR2 (cROR2) EVEVPDPNDPLGPLDGQD GPIPTLKGYFLNFLEPVNNIT IVQGQTAILHCKVAGNPPP NVRWLKNDAPVVQEPRRIII RKTEYGSRLRIQDLDTTDTG YYQCVATNGMKTITATGVL FVRLGPTHSPNHNFQDDYH EDGFCQPYRGIACARFIGNR TIYVDSLQMQGEIENRITAA FTMIGTSTHLSDQCSQFAIP SFCHFVFPLCDARSRAPKPR ELCRDECEVLESDLCRQEYTI ARSNPLILMRLQLPKCEALP MPESPDAANCMRIGIPAER LGRYHQCYNGSGTDYRGTA STTKSGHQCQPWALQHPH SHHLSSTDFPELGGGHAYC RNPGGQMEGPWCFTQNK NVRMELCDVPSCSPRDSSK MG 142 extracellular domain of murine ROR2 (mROR2) EVEDSEAIDTLGQPDGPDSP LPTLKGYFLNFLEPVNNITIV QGQTAILHCKVAGNPPPNV RWLKNDAPVVQEPRRVIIR KTEYGSRLRIQDLDTTDTGY YQCVATNGLKTITATGVLYV RLGPTHSPNHNFQDDDQE DGFCQPYRGIACARFIGNRT IYVDSLQMQGEIENRITAAF TMIGTSTQLSDQCSQFAIPS FCHFVFPLCDARSRAPKPRE LCRDECEVLENDLCRQEYTI ARSNPLILMRLQLPKCEALP MPESPDAANCMRIGIPAER LGRYHQCYNGSGADYRGM ASTTKSGHQCQPWALQHP HSHRLSSTEFPELGGGHAYC RNPGGQMEGPWCFTQNK NVRVELCDVPPCSPRDGSK MG

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PATENT LITERATURE

-   U.S. Pat. No. 5,075,109 -   U.S. Pat. No. 4,452,775 -   U.S. Pat. No. 4,667,014 -   U.S. Pat. No. 4,748,034 -   U.S. Pat. No. 5,239,660 -   U.S. Pat. No. 3,832,253 -   U.S. Pat. No. 3,854,480 -   U.S. Pat. No. 4,458,066. -   U.S. Pat. No. 8,916,159 -   WO 2010/070263 A1 -   WO 2014/013026 A1 -   WO 2014/140317 A1 -   WO 2016/102679 A1 -   WO 2013/103637 A1 -   WO 2016/142768 A1 -   WO 2014/140317 A1 

What is claimed is:
 1. A fully human antibody, or antigen-binding fragment thereof, which specifically binds to the extracellular domain of receptor tyrosine kinase-like orphan receptor 2 (ROR2) which comprises one of the following CDR sets: a) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 38, 39 and 40, respectively, and light chain CDRs 1 and 3 as set forth in SEQ ID NOs 29 and 41, respectively, with light chain CDR 2 having the sequence KAS; b) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 26-28, respectively, and light chain CDRs 1-3 as set forth in SEQ ID NOs 29, 73 and 30, respectively; c) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 94, 32 and 44, respectively, and light chain CDRs 1 and 3 as set forth in SEQ ID NOs 29 and 42, respectively, with light chain CDR 2 having the sequence KAS; d) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 38, 46 and 52, respectively, and light chain CDRs 1 and 3 as set forth in SEQ ID NOs 50 and 53, respectively, with light chain CDR 2 having the sequence AAS; e) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 38, 54 and 55, respectively, and light chain CDRs 1 and 3 as set forth in SEQ ID NOs 56 and 57, respectively, with light chain CDR 2 having the sequence LGS; f) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 58-60, respectively, and light chain CDRs 1 and 3 as set forth in SEQ ID NOs 61, and 62, respectively, with light chain CDR 2 having the sequence KAS, g) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 63-65, respectively, with light chain CDRs 1 and 3 as set forth in SEQ ID NOs 50 and 66, respectively, with light chain CDR 2 having the sequence AAS.
 2. A fully human antibody, or antigen-binding fragment thereof, which specifically binds to the extracellular domain of receptor tyrosine kinase-like orphan receptor 2 (ROR2), wherein said antibody comprises an Ig heavy chain variable region sequence and an Ig light chain variable region sequence, respectively, shown in: (i) SEQ ID NO:2 and SEQ ID NO:3; (ii) SEQ ID NO:4 and SEQ ID NO:5; (iii) SEQ ID NO:6 and SEQ ID NO:7; (iv) SEQ ID NO:8 and SEQ ID NO:9; (v) SEQ ID NO:10 and SEQ ID NO:11; (vi) SEQ ID NO:12 and SEQ ID NO:13; (vii) SEQ ID NO:14 and SEQ ID NO:15; (vii) SEQ ID NO:16 and SEQ ID NO:17; (ix) SEQ ID NO:18 and SEQ ID NO:19; (x) SEQ ID NO:20 and SEQ ID NO:21; (xi) SEQ ID NO:22 and SEQ ID NO:23; or (xii) SEQ ID NO:24 and SEQ ID NO:25.
 3. The antibody, or antigen-binding fragment thereof, according to claim 1, which comprises one of the following sequence pairs: a) the heavy chain variable region sequence of antibody GK-5A1 shown in SEQ ID NO. 8 and the light chain variable region sequence of antibody GK-5A1 shown in SEQ ID NO. 9, b) the heavy chain variable region sequence of antibody MK-3B12 shown in SEQ ID NO. 2 and the light chain variable region sequence of antibody MK-3B12 shown in SEQ ID NO. 3, c) the heavy chain variable region sequence of antibody GK-5E1 shown in SEQ ID NO. 12 and the light chain variable region sequence of antibody GK-5E1 shown in SEQ ID NO. 13, d) the heavy chain variable region sequence of antibody GK-21D3 shown in SEQ ID NO. 18 and the light chain variable region sequence of antibody GK-5E1 shown in SEQ ID NO. 19, e) the heavy chain variable region sequence of antibody MK-24C10 shown in SEQ ID NO. 20 and the light chain variable region sequence of antibody MK-24C10 shown in SEQ ID NO. 21, f) the heavy chain variable region sequence of antibody MK-24F9 shown in SEQ ID NO. 22 and the light chain variable region sequence of antibody MK-24F9 shown in SEQ ID NO. 23, g) the heavy chain variable region sequence of antibody GK-22G12 shown in SEQ ID NO. 24 and the light chain variable region sequence of antibody GK-22G12 shown in SEQ ID NO.
 25. 4. A bi- or multispecific which comprises a first antigen-binding domain that comprises an antibody, or antigen-binding fragment thereof, according to claim 1, which binds to the extracellular domain of receptor tyrosine kinase-like orphan receptor 2 (ROR2), and at least a second antigen-binding domain that binds to an effector antigen selected from the group consisting of CD3, CD16, NKG2D, NKp46, CD2, CD28 and/or CD25.
 5. A method of killing or inhibiting the growth of a cell expressing ROR2 in vitro, which method comprises administering to the cell a pharmaceutically effective amount or dose of the antibody, or antigen-binding fragment thereof, according to claim
 1. 